<XBRL>
<?xml version="1.0" encoding="us-ascii"?>
<!-- Created by Merrill Corporation -->
<!--XBRL document created with Merrill Bridge  7.3.329.0 -->
<!--Based on XBRL 2.1-->
<!--Created on: 2/26/2018 12:11:47 PM-->
<!--Modified on: 2/26/2018 12:11:47 PM-->
<link:linkbase xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd" xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xmlns:xbrli="http://www.xbrl.org/2003/instance" xmlns:xlink="http://www.w3.org/1999/xlink">
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#StatementConsolidatedStatementOfChangesInStockholdersEquity" roleURI="http://www.2u.com/role/StatementConsolidatedStatementOfChangesInStockholdersEquity" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureStockBasedCompensationTables" roleURI="http://www.2u.com/role/DisclosureStockBasedCompensationTables" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureSignificantAccountingPoliciesDetails" roleURI="http://www.2u.com/role/DisclosureSignificantAccountingPoliciesDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureBusinessCombinationEstimatedFairValuesOfAssetsAcquiredAndLiabilitiesAssumedDetails" roleURI="http://www.2u.com/role/DisclosureBusinessCombinationEstimatedFairValuesOfAssetsAcquiredAndLiabilitiesAssumedDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureBusinessCombinationEstimatedAverageUsefulLifeDetails" roleURI="http://www.2u.com/role/DisclosureBusinessCombinationEstimatedAverageUsefulLifeDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosurePropertyAndEquipmentNetDetails" roleURI="http://www.2u.com/role/DisclosurePropertyAndEquipmentNetDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureGoodwillAndAmortizableIntangibleAssetsDetails" roleURI="http://www.2u.com/role/DisclosureGoodwillAndAmortizableIntangibleAssetsDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureGoodwillAndAmortizableIntangibleAssetsEstimatedFutureAmortizationExpenseAndLicenseAgreementDetails" roleURI="http://www.2u.com/role/DisclosureGoodwillAndAmortizableIntangibleAssetsEstimatedFutureAmortizationExpenseAndLicenseAgreementDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureOperatingLeasesDetails" roleURI="http://www.2u.com/role/DisclosureOperatingLeasesDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureDebtDetails" roleURI="http://www.2u.com/role/DisclosureDebtDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureIncomeTaxesChangeInDeferredTaxValuationAllowanceDetails" roleURI="http://www.2u.com/role/DisclosureIncomeTaxesChangeInDeferredTaxValuationAllowanceDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureIncomeTaxesCarryforwardsDetails" roleURI="http://www.2u.com/role/DisclosureIncomeTaxesCarryforwardsDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureStockholdersEquityDetails" roleURI="http://www.2u.com/role/DisclosureStockholdersEquityDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureStockBasedCompensationExpenseDetails" roleURI="http://www.2u.com/role/DisclosureStockBasedCompensationExpenseDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureStockBasedCompensationOtherDetails" roleURI="http://www.2u.com/role/DisclosureStockBasedCompensationOtherDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureStockBasedCompensationEmployeeStockPurchasePlanDetails" roleURI="http://www.2u.com/role/DisclosureStockBasedCompensationEmployeeStockPurchasePlanDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureNetLossPerShareAntidilutiveDetails" roleURI="http://www.2u.com/role/DisclosureNetLossPerShareAntidilutiveDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureSegmentAndGeographicInformationConcentrationRiskDetails" roleURI="http://www.2u.com/role/DisclosureSegmentAndGeographicInformationConcentrationRiskDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureSegmentAndGeographicInformationSegmentPerformanceDetails" roleURI="http://www.2u.com/role/DisclosureSegmentAndGeographicInformationSegmentPerformanceDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureSegmentAndGeographicInformationGeographicalInformationDetails" roleURI="http://www.2u.com/role/DisclosureSegmentAndGeographicInformationGeographicalInformationDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <roleRef xlink:type="simple" xlink:href="twou-20171231.xsd#DisclosureRelatedPartyTransactionsDetails" roleURI="http://www.2u.com/role/DisclosureRelatedPartyTransactionsDetails" xmlns="http://www.xbrl.org/2003/linkbase" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/all" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension" />
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default" />
  <definitionLink xlink:role="http://www.2u.com/role/StatementConsolidatedStatementOfChangesInStockholdersEquity" xlink:type="extended" xlink:title="00300 - Statement - Consolidated Statement of Changes in Stockholders' Equity" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementEquityComponentsAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain_636552439053252657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain_636552439053252657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_CommonStockMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AdditionalPaidInCapitalMember" xlink:label="us-gaap_AdditionalPaidInCapitalMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AdditionalPaidInCapitalMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RetainedEarningsMember" xlink:label="us-gaap_RetainedEarningsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_RetainedEarningsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember" xlink:label="us-gaap_AccumulatedOtherComprehensiveIncomeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AccumulatedOtherComprehensiveIncomeMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:label="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StockholdersEquity" xlink:label="us-gaap_StockholdersEquity" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockholdersEquity" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_CommonStockSharesOutstanding" xlink:label="us-gaap_CommonStockSharesOutstanding" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_CommonStockSharesOutstanding" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_CumulativeEffectOfNewAccountingPrincipleInPeriodOfAdoptionDuration" xlink:label="twou_CumulativeEffectOfNewAccountingPrincipleInPeriodOfAdoptionDuration" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="twou_CumulativeEffectOfNewAccountingPrincipleInPeriodOfAdoptionDuration" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" xlink:to="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RestrictedStockValueSharesIssuedNetOfTaxWithholdings" xlink:label="us-gaap_RestrictedStockValueSharesIssuedNetOfTaxWithholdings" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_RestrictedStockValueSharesIssuedNetOfTaxWithholdings" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RestrictedStockSharesIssuedNetOfSharesForTaxWithholdings" xlink:label="us-gaap_RestrictedStockSharesIssuedNetOfSharesForTaxWithholdings" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_RestrictedStockValueSharesIssuedNetOfTaxWithholdings" xlink:to="us-gaap_RestrictedStockSharesIssuedNetOfSharesForTaxWithholdings" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodValueNewIssues" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodValueNewIssues" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StockGrantedDuringPeriodValueSharebasedCompensation" xlink:label="us-gaap_StockGrantedDuringPeriodValueSharebasedCompensation" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockGrantedDuringPeriodValueSharebasedCompensation" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriod" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_StockGrantedDuringPeriodValueSharebasedCompensation" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriod" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_NetIncomeLoss" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_OtherComprehensiveIncomeLossForeignCurrencyTransactionAndTranslationAdjustmentNetOfTax" xlink:label="us-gaap_OtherComprehensiveIncomeLossForeignCurrencyTransactionAndTranslationAdjustmentNetOfTax" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_OtherComprehensiveIncomeLossForeignCurrencyTransactionAndTranslationAdjustmentNetOfTax" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureStockBasedCompensationTables" xlink:type="extended" xlink:title="31103 - Disclosure - Stock-Based Compensation (Tables)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_636552439053252657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_636552439053252657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTextBlock" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTextBlock" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTextBlock" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfShareBasedPaymentAwardStockOptionsValuationAssumptionsTableTextBlock" xlink:label="us-gaap_ScheduleOfShareBasedPaymentAwardStockOptionsValuationAssumptionsTableTextBlock" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedPaymentAwardStockOptionsValuationAssumptionsTableTextBlock" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationStockOptionsActivityTableTextBlock" xlink:label="us-gaap_ScheduleOfShareBasedCompensationStockOptionsActivityTableTextBlock" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationStockOptionsActivityTableTextBlock" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationRestrictedStockUnitsAwardActivityTableTextBlock" xlink:label="us-gaap_ScheduleOfShareBasedCompensationRestrictedStockUnitsAwardActivityTableTextBlock" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationRestrictedStockUnitsAwardActivityTableTextBlock" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureSignificantAccountingPoliciesDetails" xlink:type="extended" xlink:title="40201 - Disclosure - Significant Accounting Policies (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:label="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:to="us-gaap_PropertyPlantAndEquipmentByTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain_636552439053252657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain_636552439053252657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ComputerEquipmentMember" xlink:label="us-gaap_ComputerEquipmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_ComputerEquipmentMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FurnitureAndFixturesMember" xlink:label="us-gaap_FurnitureAndFixturesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_FurnitureAndFixturesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_LeaseholdImprovementsMember" xlink:label="us-gaap_LeaseholdImprovementsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_LeaseholdImprovementsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SoftwareDevelopmentMember" xlink:label="us-gaap_SoftwareDevelopmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_SoftwareDevelopmentMember" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_LandlordFundedLeaseholdImprovementsMember" xlink:label="twou_LandlordFundedLeaseholdImprovementsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="twou_LandlordFundedLeaseholdImprovementsMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StatementBusinessSegmentsAxis" xlink:label="us-gaap_StatementBusinessSegmentsAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:to="us-gaap_StatementBusinessSegmentsAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SegmentDomain" xlink:label="us-gaap_SegmentDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementBusinessSegmentsAxis" xlink:to="us-gaap_SegmentDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SegmentDomain" xlink:label="us-gaap_SegmentDomain_636552439053252657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_StatementBusinessSegmentsAxis" xlink:to="us-gaap_SegmentDomain_636552439053252657" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_GraduateProgramSegmentMember" xlink:label="twou_GraduateProgramSegmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SegmentDomain" xlink:to="twou_GraduateProgramSegmentMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_ShortCourseSegmentMember" xlink:label="twou_ShortCourseSegmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_SegmentDomain" xlink:to="twou_ShortCourseSegmentMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AdjustmentsForNewAccountingPronouncementsAxis" xlink:label="us-gaap_AdjustmentsForNewAccountingPronouncementsAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:to="us-gaap_AdjustmentsForNewAccountingPronouncementsAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_TypeOfAdoptionMember" xlink:label="us-gaap_TypeOfAdoptionMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_AdjustmentsForNewAccountingPronouncementsAxis" xlink:to="us-gaap_TypeOfAdoptionMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_TypeOfAdoptionMember" xlink:label="us-gaap_TypeOfAdoptionMember_636552439053252657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_AdjustmentsForNewAccountingPronouncementsAxis" xlink:to="us-gaap_TypeOfAdoptionMember_636552439053252657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AccountingStandardsUpdate201609Member" xlink:label="us-gaap_AccountingStandardsUpdate201609Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_TypeOfAdoptionMember" xlink:to="us-gaap_AccountingStandardsUpdate201609Member" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RangeAxis" xlink:label="us-gaap_RangeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="4" xlink:from="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:to="us-gaap_RangeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RangeMember" xlink:label="us-gaap_RangeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_RangeAxis" xlink:to="us-gaap_RangeMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RangeMember" xlink:label="us-gaap_RangeMember_636552439053252657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_RangeAxis" xlink:to="us-gaap_RangeMember_636552439053252657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_MinimumMember" xlink:label="us-gaap_MinimumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_RangeMember" xlink:to="us-gaap_MinimumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_MaximumMember" xlink:label="us-gaap_MaximumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_RangeMember" xlink:to="us-gaap_MaximumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BalanceSheetLocationAxis" xlink:label="us-gaap_BalanceSheetLocationAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="5" xlink:from="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:to="us-gaap_BalanceSheetLocationAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BalanceSheetLocationDomain" xlink:label="us-gaap_BalanceSheetLocationDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_BalanceSheetLocationAxis" xlink:to="us-gaap_BalanceSheetLocationDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BalanceSheetLocationDomain" xlink:label="us-gaap_BalanceSheetLocationDomain_636552439053252657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_BalanceSheetLocationAxis" xlink:to="us-gaap_BalanceSheetLocationDomain_636552439053252657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AccountsPayableAndAccruedLiabilitiesMember" xlink:label="us-gaap_AccountsPayableAndAccruedLiabilitiesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_BalanceSheetLocationDomain" xlink:to="us-gaap_AccountsPayableAndAccruedLiabilitiesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PropertyPlantAndEquipmentLineItems" xlink:label="us-gaap_PropertyPlantAndEquipmentLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PropertyPlantAndEquipmentUsefulLife" xlink:label="us-gaap_PropertyPlantAndEquipmentUsefulLife" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentUsefulLife" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsNetAbstract" xlink:label="us-gaap_FiniteLivedIntangibleAssetsNetAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetsNetAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetUsefulLife" xlink:label="us-gaap_FiniteLivedIntangibleAssetUsefulLife" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_FiniteLivedIntangibleAssetsNetAbstract" xlink:to="us-gaap_FiniteLivedIntangibleAssetUsefulLife" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_CapitalizedContentDevelopmentCostsUsefulLife" xlink:label="twou_CapitalizedContentDevelopmentCostsUsefulLife" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_FiniteLivedIntangibleAssetsNetAbstract" xlink:to="twou_CapitalizedContentDevelopmentCostsUsefulLife" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_CashFlowNoncashInvestingAndFinancingActivitiesDisclosureAbstract" xlink:label="us-gaap_CashFlowNoncashInvestingAndFinancingActivitiesDisclosureAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_CashFlowNoncashInvestingAndFinancingActivitiesDisclosureAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PropertyPlantAndEquipmentAdditions" xlink:label="us-gaap_PropertyPlantAndEquipmentAdditions" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_CashFlowNoncashInvestingAndFinancingActivitiesDisclosureAbstract" xlink:to="us-gaap_PropertyPlantAndEquipmentAdditions" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_NonCashCapitalExpenditure" xlink:label="twou_NonCashCapitalExpenditure" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_CashFlowNoncashInvestingAndFinancingActivitiesDisclosureAbstract" xlink:to="twou_NonCashCapitalExpenditure" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SegmentReportingInformationRevenueAbstract" xlink:label="us-gaap_SegmentReportingInformationRevenueAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_SegmentReportingInformationRevenueAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_TermOfRevenueContracts" xlink:label="twou_TermOfRevenueContracts" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SegmentReportingInformationRevenueAbstract" xlink:to="twou_TermOfRevenueContracts" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AllowanceForDoubtfulAccountsReceivable" xlink:label="us-gaap_AllowanceForDoubtfulAccountsReceivable" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_SegmentReportingInformationRevenueAbstract" xlink:to="us-gaap_AllowanceForDoubtfulAccountsReceivable" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_MarketingAndSalesCostsAbstract" xlink:label="twou_MarketingAndSalesCostsAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="twou_MarketingAndSalesCostsAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AccruedMarketingCostsCurrent" xlink:label="us-gaap_AccruedMarketingCostsCurrent" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="twou_MarketingAndSalesCostsAbstract" xlink:to="us-gaap_AccruedMarketingCostsCurrent" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_NewAccountingPronouncementOrChangeInAccountingPrincipleRetrospectiveAdjustmentsAbstract" xlink:label="us-gaap_NewAccountingPronouncementOrChangeInAccountingPrincipleRetrospectiveAdjustmentsAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_NewAccountingPronouncementOrChangeInAccountingPrincipleRetrospectiveAdjustmentsAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_NewAccountingPronouncementOrChangeInAccountingPrincipleCumulativeEffectOfChangeOnEquityOrNetAssets1" xlink:label="us-gaap_NewAccountingPronouncementOrChangeInAccountingPrincipleCumulativeEffectOfChangeOnEquityOrNetAssets1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_NewAccountingPronouncementOrChangeInAccountingPrincipleRetrospectiveAdjustmentsAbstract" xlink:to="us-gaap_NewAccountingPronouncementOrChangeInAccountingPrincipleCumulativeEffectOfChangeOnEquityOrNetAssets1" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureBusinessCombinationEstimatedFairValuesOfAssetsAcquiredAndLiabilitiesAssumedDetails" xlink:type="extended" xlink:title="40301 - Disclosure - Business Combination - Estimated fair values of the assets acquired and liabilities assumed (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable" xlink:label="us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BusinessAcquisitionAxis" xlink:label="us-gaap_BusinessAcquisitionAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable" xlink:to="us-gaap_BusinessAcquisitionAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BusinessAcquisitionAcquireeDomain" xlink:label="us-gaap_BusinessAcquisitionAcquireeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_BusinessAcquisitionAxis" xlink:to="us-gaap_BusinessAcquisitionAcquireeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BusinessAcquisitionAcquireeDomain" xlink:label="us-gaap_BusinessAcquisitionAcquireeDomain_636552439053252657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_BusinessAcquisitionAxis" xlink:to="us-gaap_BusinessAcquisitionAcquireeDomain_636552439053252657" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_GetEducatedInternationalProprietaryLimitedMember" xlink:label="twou_GetEducatedInternationalProprietaryLimitedMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_BusinessAcquisitionAcquireeDomain" xlink:to="twou_GetEducatedInternationalProprietaryLimitedMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" xlink:label="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable" xlink:to="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:label="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" xlink:to="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:label="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_636552439053252657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" xlink:to="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain_636552439053252657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_DevelopedTechnologyRightsMember" xlink:label="us-gaap_DevelopedTechnologyRightsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="us-gaap_DevelopedTechnologyRightsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ArtisticRelatedIntangibleAssetsMember" xlink:label="us-gaap_ArtisticRelatedIntangibleAssetsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="us-gaap_ArtisticRelatedIntangibleAssetsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ContractualRightsMember" xlink:label="us-gaap_ContractualRightsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="us-gaap_ContractualRightsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_TradeNamesMember" xlink:label="us-gaap_TradeNamesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="us-gaap_TradeNamesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RangeAxis" xlink:label="us-gaap_RangeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable" xlink:to="us-gaap_RangeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RangeMember" xlink:label="us-gaap_RangeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_RangeAxis" xlink:to="us-gaap_RangeMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_MinimumMember" xlink:label="us-gaap_MinimumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_RangeMember" xlink:to="us-gaap_MinimumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_MaximumMember" xlink:label="us-gaap_MaximumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_RangeMember" xlink:to="us-gaap_MaximumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BusinessAcquisitionLineItems" xlink:label="us-gaap_BusinessAcquisitionLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_BusinessAcquisitionLineItems" xlink:to="us-gaap_ScheduleOfBusinessAcquisitionsByAcquisitionTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PaymentsToAcquireBusinessesGross" xlink:label="us-gaap_PaymentsToAcquireBusinessesGross" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_BusinessAcquisitionLineItems" xlink:to="us-gaap_PaymentsToAcquireBusinessesGross" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BusinessCombinationContingentConsiderationArrangementsRangeOfOutcomesValueHigh" xlink:label="us-gaap_BusinessCombinationContingentConsiderationArrangementsRangeOfOutcomesValueHigh" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_BusinessAcquisitionLineItems" xlink:to="us-gaap_BusinessCombinationContingentConsiderationArrangementsRangeOfOutcomesValueHigh" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:label="us-gaap_CommonStockParOrStatedValuePerShare" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_BusinessAcquisitionLineItems" xlink:to="us-gaap_CommonStockParOrStatedValuePerShare" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_BusinessAcquisitionLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedCashAndEquivalents" xlink:label="us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedCashAndEquivalents" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_BusinessAcquisitionLineItems" xlink:to="us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedCashAndEquivalents" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedCurrentAssets" xlink:label="us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedCurrentAssets" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_BusinessAcquisitionLineItems" xlink:to="us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedCurrentAssets" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedPropertyPlantAndEquipment" xlink:label="us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedPropertyPlantAndEquipment" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_BusinessAcquisitionLineItems" xlink:to="us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedPropertyPlantAndEquipment" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedIntangibles" xlink:label="us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedIntangibles" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_BusinessAcquisitionLineItems" xlink:to="us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedIntangibles" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_Goodwill" xlink:label="us-gaap_Goodwill" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="us-gaap_BusinessAcquisitionLineItems" xlink:to="us-gaap_Goodwill" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedCurrentLiabilities" xlink:label="us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedCurrentLiabilities" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="us-gaap_BusinessAcquisitionLineItems" xlink:to="us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedCurrentLiabilities" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedLiabilities" xlink:label="us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedLiabilities" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="12" xlink:from="us-gaap_BusinessAcquisitionLineItems" xlink:to="us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredAndLiabilitiesAssumedLiabilities" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredGoodwillAndLiabilitiesAssumedNet" xlink:label="us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredGoodwillAndLiabilitiesAssumedNet" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="13" xlink:from="us-gaap_BusinessAcquisitionLineItems" xlink:to="us-gaap_BusinessCombinationRecognizedIdentifiableAssetsAcquiredGoodwillAndLiabilitiesAssumedNet" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_GoodwillDecreaseInPeriodOffsettingInOtherCurrentLiabilitiesRelatedToContingentConsideration" xlink:label="twou_GoodwillDecreaseInPeriodOffsettingInOtherCurrentLiabilitiesRelatedToContingentConsideration" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="14" xlink:from="us-gaap_BusinessAcquisitionLineItems" xlink:to="twou_GoodwillDecreaseInPeriodOffsettingInOtherCurrentLiabilitiesRelatedToContingentConsideration" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureBusinessCombinationEstimatedAverageUsefulLifeDetails" xlink:type="extended" xlink:title="40302 - Disclosure - Business Combination - Estimated Average Useful Life (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfAcquiredFiniteLivedIntangibleAssetByMajorClassTable" xlink:label="us-gaap_ScheduleOfAcquiredFiniteLivedIntangibleAssetByMajorClassTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BusinessAcquisitionAxis" xlink:label="us-gaap_BusinessAcquisitionAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfAcquiredFiniteLivedIntangibleAssetByMajorClassTable" xlink:to="us-gaap_BusinessAcquisitionAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BusinessAcquisitionAcquireeDomain" xlink:label="us-gaap_BusinessAcquisitionAcquireeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_BusinessAcquisitionAxis" xlink:to="us-gaap_BusinessAcquisitionAcquireeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_GetEducatedInternationalProprietaryLimitedMember" xlink:label="twou_GetEducatedInternationalProprietaryLimitedMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_BusinessAcquisitionAcquireeDomain" xlink:to="twou_GetEducatedInternationalProprietaryLimitedMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" xlink:label="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfAcquiredFiniteLivedIntangibleAssetByMajorClassTable" xlink:to="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:label="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" xlink:to="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_DevelopedTechnologyRightsMember" xlink:label="us-gaap_DevelopedTechnologyRightsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="us-gaap_DevelopedTechnologyRightsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ArtisticRelatedIntangibleAssetsMember" xlink:label="us-gaap_ArtisticRelatedIntangibleAssetsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="us-gaap_ArtisticRelatedIntangibleAssetsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ContractualRightsMember" xlink:label="us-gaap_ContractualRightsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="us-gaap_ContractualRightsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_TradeNamesMember" xlink:label="us-gaap_TradeNamesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="us-gaap_TradeNamesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AcquiredFiniteLivedIntangibleAssetsLineItems" xlink:label="us-gaap_AcquiredFiniteLivedIntangibleAssetsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_AcquiredFiniteLivedIntangibleAssetsLineItems" xlink:to="us-gaap_ScheduleOfAcquiredFiniteLivedIntangibleAssetByMajorClassTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetUsefulLife" xlink:label="us-gaap_FiniteLivedIntangibleAssetUsefulLife" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_AcquiredFiniteLivedIntangibleAssetsLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetUsefulLife" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosurePropertyAndEquipmentNetDetails" xlink:type="extended" xlink:title="40401 - Disclosure - Property and Equipment, Net (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:label="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:to="us-gaap_PropertyPlantAndEquipmentByTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ComputerEquipmentMember" xlink:label="us-gaap_ComputerEquipmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_ComputerEquipmentMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FurnitureAndFixturesMember" xlink:label="us-gaap_FurnitureAndFixturesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_FurnitureAndFixturesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_LeaseholdImprovementsMember" xlink:label="us-gaap_LeaseholdImprovementsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_LeaseholdImprovementsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_LeaseholdImprovementsInProcessMember" xlink:label="twou_LeaseholdImprovementsInProcessMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="twou_LeaseholdImprovementsInProcessMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PropertyPlantAndEquipmentLineItems" xlink:label="us-gaap_PropertyPlantAndEquipmentLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PropertyPlantAndEquipmentGross" xlink:label="us-gaap_PropertyPlantAndEquipmentGross" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentGross" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment" xlink:label="us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PropertyPlantAndEquipmentNet" xlink:label="us-gaap_PropertyPlantAndEquipmentNet" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentNet" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_Depreciation" xlink:label="us-gaap_Depreciation" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_Depreciation" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureGoodwillAndAmortizableIntangibleAssetsDetails" xlink:type="extended" xlink:title="40501 - Disclosure - Goodwill and Amortizable Intangible Assets (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable" xlink:label="us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BusinessAcquisitionAxis" xlink:label="us-gaap_BusinessAcquisitionAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable" xlink:to="us-gaap_BusinessAcquisitionAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BusinessAcquisitionAcquireeDomain" xlink:label="us-gaap_BusinessAcquisitionAcquireeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_BusinessAcquisitionAxis" xlink:to="us-gaap_BusinessAcquisitionAcquireeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_GetEducatedInternationalProprietaryLimitedMember" xlink:label="twou_GetEducatedInternationalProprietaryLimitedMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_BusinessAcquisitionAcquireeDomain" xlink:to="twou_GetEducatedInternationalProprietaryLimitedMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StatementBusinessSegmentsAxis" xlink:label="us-gaap_StatementBusinessSegmentsAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable" xlink:to="us-gaap_StatementBusinessSegmentsAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SegmentDomain" xlink:label="us-gaap_SegmentDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementBusinessSegmentsAxis" xlink:to="us-gaap_SegmentDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_ShortCourseSegmentMember" xlink:label="twou_ShortCourseSegmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SegmentDomain" xlink:to="twou_ShortCourseSegmentMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" xlink:label="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable" xlink:to="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:label="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" xlink:to="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_DevelopedTechnologyRightsMember" xlink:label="us-gaap_DevelopedTechnologyRightsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="us-gaap_DevelopedTechnologyRightsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ArtisticRelatedIntangibleAssetsMember" xlink:label="us-gaap_ArtisticRelatedIntangibleAssetsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="us-gaap_ArtisticRelatedIntangibleAssetsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ContractualRightsMember" xlink:label="us-gaap_ContractualRightsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="us-gaap_ContractualRightsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_TradeNamesMember" xlink:label="us-gaap_TradeNamesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="us-gaap_TradeNamesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_InProcessCapitalizedTechnologyAndContentDevelopmentMember" xlink:label="twou_InProcessCapitalizedTechnologyAndContentDevelopmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="twou_InProcessCapitalizedTechnologyAndContentDevelopmentMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AcquiredFiniteLivedIntangibleAssetsLineItems" xlink:label="us-gaap_AcquiredFiniteLivedIntangibleAssetsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_AcquiredFiniteLivedIntangibleAssetsLineItems" xlink:to="us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_Goodwill" xlink:label="us-gaap_Goodwill" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_AcquiredFiniteLivedIntangibleAssetsLineItems" xlink:to="us-gaap_Goodwill" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_GoodwillForeignCurrencyTranslationGainLoss" xlink:label="us-gaap_GoodwillForeignCurrencyTranslationGainLoss" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_AcquiredFiniteLivedIntangibleAssetsLineItems" xlink:to="us-gaap_GoodwillForeignCurrencyTranslationGainLoss" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetUsefulLife" xlink:label="us-gaap_FiniteLivedIntangibleAssetUsefulLife" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_AcquiredFiniteLivedIntangibleAssetsLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetUsefulLife" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsGross" xlink:label="us-gaap_FiniteLivedIntangibleAssetsGross" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_AcquiredFiniteLivedIntangibleAssetsLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetsGross" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsAccumulatedAmortization" xlink:label="us-gaap_FiniteLivedIntangibleAssetsAccumulatedAmortization" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_AcquiredFiniteLivedIntangibleAssetsLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetsAccumulatedAmortization" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsNet" xlink:label="us-gaap_FiniteLivedIntangibleAssetsNet" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_AcquiredFiniteLivedIntangibleAssetsLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetsNet" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AmortizationOfIntangibleAssets" xlink:label="us-gaap_AmortizationOfIntangibleAssets" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_AcquiredFiniteLivedIntangibleAssetsLineItems" xlink:to="us-gaap_AmortizationOfIntangibleAssets" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureGoodwillAndAmortizableIntangibleAssetsEstimatedFutureAmortizationExpenseAndLicenseAgreementDetails" xlink:type="extended" xlink:title="40502 - Disclosure - Goodwill and Amortizable Intangible Assets - Estimated Future Amortization Expense and License agreement (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable" xlink:label="us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_CounterpartyNameAxis" xlink:label="us-gaap_CounterpartyNameAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable" xlink:to="us-gaap_CounterpartyNameAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RepurchaseAgreementCounterpartyNameDomain" xlink:label="us-gaap_RepurchaseAgreementCounterpartyNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_CounterpartyNameAxis" xlink:to="us-gaap_RepurchaseAgreementCounterpartyNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RepurchaseAgreementCounterpartyNameDomain" xlink:label="us-gaap_RepurchaseAgreementCounterpartyNameDomain_636552439053252657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_CounterpartyNameAxis" xlink:to="us-gaap_RepurchaseAgreementCounterpartyNameDomain_636552439053252657" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_FlatironSchoolIncMember" xlink:label="twou_FlatironSchoolIncMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_RepurchaseAgreementCounterpartyNameDomain" xlink:to="twou_FlatironSchoolIncMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable" xlink:to="us-gaap_TypeOfArrangementAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_636552439053252657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_636552439053252657" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_SoftwareLicenseAndServicesAgreementMember" xlink:label="twou_SoftwareLicenseAndServicesAgreementMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="twou_SoftwareLicenseAndServicesAgreementMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" xlink:label="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable" xlink:to="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:label="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_FiniteLivedIntangibleAssetsByMajorClassAxis" xlink:to="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_ExcludingInProcessCapitalizedTechnologyAndContentDevelopmentMember" xlink:label="twou_ExcludingInProcessCapitalizedTechnologyAndContentDevelopmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_FiniteLivedIntangibleAssetsMajorClassNameDomain" xlink:to="twou_ExcludingInProcessCapitalizedTechnologyAndContentDevelopmentMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsLineItems" xlink:label="us-gaap_FiniteLivedIntangibleAssetsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_FiniteLivedIntangibleAssetsLineItems" xlink:to="us-gaap_ScheduleOfFiniteLivedIntangibleAssetsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsFutureAmortizationExpenseAbstract" xlink:label="us-gaap_FiniteLivedIntangibleAssetsFutureAmortizationExpenseAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_FiniteLivedIntangibleAssetsLineItems" xlink:to="us-gaap_FiniteLivedIntangibleAssetsFutureAmortizationExpenseAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseNextTwelveMonths" xlink:label="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseNextTwelveMonths" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_FiniteLivedIntangibleAssetsFutureAmortizationExpenseAbstract" xlink:to="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseNextTwelveMonths" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearTwo" xlink:label="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearTwo" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_FiniteLivedIntangibleAssetsFutureAmortizationExpenseAbstract" xlink:to="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearTwo" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearThree" xlink:label="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearThree" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_FiniteLivedIntangibleAssetsFutureAmortizationExpenseAbstract" xlink:to="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearThree" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearFour" xlink:label="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearFour" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_FiniteLivedIntangibleAssetsFutureAmortizationExpenseAbstract" xlink:to="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearFour" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearFive" xlink:label="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearFive" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_FiniteLivedIntangibleAssetsFutureAmortizationExpenseAbstract" xlink:to="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseYearFive" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseAfterYearFive" xlink:label="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseAfterYearFive" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_FiniteLivedIntangibleAssetsFutureAmortizationExpenseAbstract" xlink:to="us-gaap_FiniteLivedIntangibleAssetsAmortizationExpenseAfterYearFive" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_FiniteLivedIntangibleAssetsNet" xlink:label="us-gaap_FiniteLivedIntangibleAssetsNet" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_FiniteLivedIntangibleAssetsFutureAmortizationExpenseAbstract" xlink:to="us-gaap_FiniteLivedIntangibleAssetsNet" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_CostOfServicesLicensesAndServices" xlink:label="us-gaap_CostOfServicesLicensesAndServices" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_FiniteLivedIntangibleAssetsLineItems" xlink:to="us-gaap_CostOfServicesLicensesAndServices" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureOperatingLeasesDetails" xlink:type="extended" xlink:title="40701 - Disclosure - Operating Leases (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfOperatingLeasedAssetsTable" xlink:label="us-gaap_ScheduleOfOperatingLeasedAssetsTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_LeaseArrangementTypeAxis" xlink:label="us-gaap_LeaseArrangementTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfOperatingLeasedAssetsTable" xlink:to="us-gaap_LeaseArrangementTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_LeaseArrangementTypeDomain" xlink:label="us-gaap_LeaseArrangementTypeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_LeaseArrangementTypeAxis" xlink:to="us-gaap_LeaseArrangementTypeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_LeaseArrangementTypeDomain" xlink:label="us-gaap_LeaseArrangementTypeDomain_636552439053408657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_LeaseArrangementTypeAxis" xlink:to="us-gaap_LeaseArrangementTypeDomain_636552439053408657" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_LeasedFacilityInBrooklynNewYorkMember" xlink:label="twou_LeasedFacilityInBrooklynNewYorkMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_LeaseArrangementTypeDomain" xlink:to="twou_LeasedFacilityInBrooklynNewYorkMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_OperatingLeasedAssetsLineItems" xlink:label="us-gaap_OperatingLeasedAssetsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_OperatingLeasedAssetsLineItems" xlink:to="us-gaap_ScheduleOfOperatingLeasedAssetsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_OperatingLeaseNumberOfFloorsLeased" xlink:label="twou_OperatingLeaseNumberOfFloorsLeased" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_OperatingLeasedAssetsLineItems" xlink:to="twou_OperatingLeaseNumberOfFloorsLeased" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AreaOfRealEstateProperty" xlink:label="us-gaap_AreaOfRealEstateProperty" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_OperatingLeasedAssetsLineItems" xlink:to="us-gaap_AreaOfRealEstateProperty" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_OperatingLeasesFutureMinimumPaymentsDue" xlink:label="us-gaap_OperatingLeasesFutureMinimumPaymentsDue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_OperatingLeasedAssetsLineItems" xlink:to="us-gaap_OperatingLeasesFutureMinimumPaymentsDue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_OperatingLeaseLeaseTerm" xlink:label="twou_OperatingLeaseLeaseTerm" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_OperatingLeasedAssetsLineItems" xlink:to="twou_OperatingLeaseLeaseTerm" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_OperatingLeasesFutureMinimumPaymentsDueAbstract" xlink:label="us-gaap_OperatingLeasesFutureMinimumPaymentsDueAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_OperatingLeasedAssetsLineItems" xlink:to="us-gaap_OperatingLeasesFutureMinimumPaymentsDueAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_OperatingLeasesFutureMinimumPaymentsRemainderOfFiscalYear" xlink:label="us-gaap_OperatingLeasesFutureMinimumPaymentsRemainderOfFiscalYear" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_OperatingLeasesFutureMinimumPaymentsDueAbstract" xlink:to="us-gaap_OperatingLeasesFutureMinimumPaymentsRemainderOfFiscalYear" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_OperatingLeasesFutureMinimumPaymentsDueInTwoYears" xlink:label="us-gaap_OperatingLeasesFutureMinimumPaymentsDueInTwoYears" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_OperatingLeasesFutureMinimumPaymentsDueAbstract" xlink:to="us-gaap_OperatingLeasesFutureMinimumPaymentsDueInTwoYears" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_OperatingLeasesFutureMinimumPaymentsDueInThreeYears" xlink:label="us-gaap_OperatingLeasesFutureMinimumPaymentsDueInThreeYears" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_OperatingLeasesFutureMinimumPaymentsDueAbstract" xlink:to="us-gaap_OperatingLeasesFutureMinimumPaymentsDueInThreeYears" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_OperatingLeasesFutureMinimumPaymentsDueInFourYears" xlink:label="us-gaap_OperatingLeasesFutureMinimumPaymentsDueInFourYears" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_OperatingLeasesFutureMinimumPaymentsDueAbstract" xlink:to="us-gaap_OperatingLeasesFutureMinimumPaymentsDueInFourYears" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_OperatingLeasesFutureMinimumPaymentsDueInFiveYears" xlink:label="us-gaap_OperatingLeasesFutureMinimumPaymentsDueInFiveYears" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_OperatingLeasesFutureMinimumPaymentsDueAbstract" xlink:to="us-gaap_OperatingLeasesFutureMinimumPaymentsDueInFiveYears" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_OperatingLeasesFutureMinimumPaymentsDueThereafter" xlink:label="us-gaap_OperatingLeasesFutureMinimumPaymentsDueThereafter" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_OperatingLeasesFutureMinimumPaymentsDueAbstract" xlink:to="us-gaap_OperatingLeasesFutureMinimumPaymentsDueThereafter" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_DeferredRentCredit" xlink:label="us-gaap_DeferredRentCredit" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_OperatingLeasedAssetsLineItems" xlink:to="us-gaap_DeferredRentCredit" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_OperatingLeasesRentExpenseSubleaseRentals1" xlink:label="us-gaap_OperatingLeasesRentExpenseSubleaseRentals1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_OperatingLeasedAssetsLineItems" xlink:to="us-gaap_OperatingLeasesRentExpenseSubleaseRentals1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_OperatingLeasesRentExpenseNet" xlink:label="us-gaap_OperatingLeasesRentExpenseNet" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_OperatingLeasedAssetsLineItems" xlink:to="us-gaap_OperatingLeasesRentExpenseNet" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureDebtDetails" xlink:type="extended" xlink:title="40801 - Disclosure - Debt (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.sec.gov/dei/2014/dei-2014-01-31.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.sec.gov/dei/2014/dei-2014-01-31.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.sec.gov/dei/2014/dei-2014-01-31.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain_636552439053564657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain_636552439053564657" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_HarkinsRoadLlcMember" xlink:label="twou_HarkinsRoadLlcMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="dei_EntityDomain" xlink:to="twou_HarkinsRoadLlcMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_VariableRateAxis" xlink:label="us-gaap_VariableRateAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_VariableRateAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_VariableRateDomain" xlink:label="us-gaap_VariableRateDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_VariableRateAxis" xlink:to="us-gaap_VariableRateDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_VariableRateDomain" xlink:label="us-gaap_VariableRateDomain_636552439053564657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_VariableRateAxis" xlink:to="us-gaap_VariableRateDomain_636552439053564657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_BaseRateMember" xlink:label="us-gaap_BaseRateMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_VariableRateDomain" xlink:to="us-gaap_BaseRateMember" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_FederalFundRateMember" xlink:label="twou_FederalFundRateMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_VariableRateDomain" xlink:to="twou_FederalFundRateMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_ThirtyDaysLiborMember" xlink:label="twou_ThirtyDaysLiborMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_VariableRateDomain" xlink:to="twou_ThirtyDaysLiborMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_LondonInterbankOfferedRateLIBORMember" xlink:label="us-gaap_LondonInterbankOfferedRateLIBORMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_VariableRateDomain" xlink:to="us-gaap_LondonInterbankOfferedRateLIBORMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RangeAxis" xlink:label="us-gaap_RangeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_RangeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RangeMember" xlink:label="us-gaap_RangeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_RangeAxis" xlink:to="us-gaap_RangeMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_MinimumMember" xlink:label="us-gaap_MinimumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_RangeMember" xlink:to="us-gaap_MinimumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StatementBusinessSegmentsAxis" xlink:label="us-gaap_StatementBusinessSegmentsAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="4" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementBusinessSegmentsAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SegmentDomain" xlink:label="us-gaap_SegmentDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementBusinessSegmentsAxis" xlink:to="us-gaap_SegmentDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_ShortCourseSegmentMember" xlink:label="twou_ShortCourseSegmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SegmentDomain" xlink:to="twou_ShortCourseSegmentMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_CreditFacilityAxis" xlink:label="us-gaap_CreditFacilityAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="5" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_CreditFacilityAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_CreditFacilityDomain" xlink:label="us-gaap_CreditFacilityDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_CreditFacilityAxis" xlink:to="us-gaap_CreditFacilityDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_CreditFacilityDomain" xlink:label="us-gaap_CreditFacilityDomain_636552439053564657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_CreditFacilityAxis" xlink:to="us-gaap_CreditFacilityDomain_636552439053564657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StandbyLettersOfCreditMember" xlink:label="us-gaap_StandbyLettersOfCreditMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_CreditFacilityDomain" xlink:to="us-gaap_StandbyLettersOfCreditMember" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_RevolvingWorkingCapitalFacilityMember" xlink:label="twou_RevolvingWorkingCapitalFacilityMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_CreditFacilityDomain" xlink:to="twou_RevolvingWorkingCapitalFacilityMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_LineOfCreditFacilityAxis" xlink:label="us-gaap_LineOfCreditFacilityAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="6" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_LineOfCreditFacilityAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_LineOfCreditFacilityLenderDomain" xlink:label="us-gaap_LineOfCreditFacilityLenderDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_LineOfCreditFacilityAxis" xlink:to="us-gaap_LineOfCreditFacilityLenderDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_LineOfCreditFacilityLenderDomain" xlink:label="us-gaap_LineOfCreditFacilityLenderDomain_636552439053564657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_LineOfCreditFacilityAxis" xlink:to="us-gaap_LineOfCreditFacilityLenderDomain_636552439053564657" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_PrinceGeorgesCountyMarylandMember" xlink:label="twou_PrinceGeorgesCountyMarylandMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_LineOfCreditFacilityLenderDomain" xlink:to="twou_PrinceGeorgesCountyMarylandMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_DepartmentOfCommerceMember" xlink:label="twou_DepartmentOfCommerceMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_LineOfCreditFacilityLenderDomain" xlink:to="twou_DepartmentOfCommerceMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SubsequentEventTypeAxis" xlink:label="us-gaap_SubsequentEventTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="7" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsequentEventTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain_636552439053564657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain_636552439053564657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SubsequentEventMember" xlink:label="us-gaap_SubsequentEventMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SubsequentEventTypeDomain" xlink:to="us-gaap_SubsequentEventMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_DebtInstrumentLineItems" xlink:label="us-gaap_DebtInstrumentLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_StatementTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_LineOfCreditFacilityAbstract" xlink:label="us-gaap_LineOfCreditFacilityAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_LineOfCreditFacilityAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity" xlink:label="us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_LineOfCreditFacilityAbstract" xlink:to="us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_LineOfCredit" xlink:label="us-gaap_LineOfCredit" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_LineOfCreditFacilityAbstract" xlink:to="us-gaap_LineOfCredit" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_DebtInstrumentDescriptionOfVariableRateBasis" xlink:label="us-gaap_DebtInstrumentDescriptionOfVariableRateBasis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_LineOfCreditFacilityAbstract" xlink:to="us-gaap_DebtInstrumentDescriptionOfVariableRateBasis" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_DebtInstrumentBasisSpreadOnVariableRate1" xlink:label="us-gaap_DebtInstrumentBasisSpreadOnVariableRate1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_LineOfCreditFacilityAbstract" xlink:to="us-gaap_DebtInstrumentBasisSpreadOnVariableRate1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_CovenantsRatio" xlink:label="twou_CovenantsRatio" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_LineOfCreditFacilityAbstract" xlink:to="twou_CovenantsRatio" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_AdjustedQuickRatio" xlink:label="twou_AdjustedQuickRatio" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_LineOfCreditFacilityAbstract" xlink:to="twou_AdjustedQuickRatio" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SecurityDeposit" xlink:label="us-gaap_SecurityDeposit" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_LineOfCreditFacilityAbstract" xlink:to="us-gaap_SecurityDeposit" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_LineOfCreditFacilityInterestRateDuringPeriod" xlink:label="us-gaap_LineOfCreditFacilityInterestRateDuringPeriod" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_LineOfCreditFacilityAbstract" xlink:to="us-gaap_LineOfCreditFacilityInterestRateDuringPeriod" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_GovernmentGrantsAbstract" xlink:label="twou_GovernmentGrantsAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="twou_GovernmentGrantsAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_DeferredGovernmentGrantObligationsNoncurrent" xlink:label="twou_DeferredGovernmentGrantObligationsNoncurrent" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="twou_GovernmentGrantsAbstract" xlink:to="twou_DeferredGovernmentGrantObligationsNoncurrent" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_NumberOfGovernmentGrants" xlink:label="twou_NumberOfGovernmentGrants" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="twou_GovernmentGrantsAbstract" xlink:to="twou_NumberOfGovernmentGrants" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:label="us-gaap_DebtInstrumentInterestRateStatedPercentage" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="twou_GovernmentGrantsAbstract" xlink:to="us-gaap_DebtInstrumentInterestRateStatedPercentage" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_NumberOfEmployeesToEmployDuringTermOfLoanToAvoidRepaymentOfPrincipalBalanceAndAccruedInterestOfLoan" xlink:label="twou_NumberOfEmployeesToEmployDuringTermOfLoanToAvoidRepaymentOfPrincipalBalanceAndAccruedInterestOfLoan" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="twou_GovernmentGrantsAbstract" xlink:to="twou_NumberOfEmployeesToEmployDuringTermOfLoanToAvoidRepaymentOfPrincipalBalanceAndAccruedInterestOfLoan" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_NumberOfEmployeesToEmployDuringTermOfLoanToAvoidRepaymentOfProratedPortionOfLoanAndInterest" xlink:label="twou_NumberOfEmployeesToEmployDuringTermOfLoanToAvoidRepaymentOfProratedPortionOfLoanAndInterest" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="twou_GovernmentGrantsAbstract" xlink:to="twou_NumberOfEmployeesToEmployDuringTermOfLoanToAvoidRepaymentOfProratedPortionOfLoanAndInterest" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_ProratedPortionOfLoanEmployee" xlink:label="twou_ProratedPortionOfLoanEmployee" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="twou_GovernmentGrantsAbstract" xlink:to="twou_ProratedPortionOfLoanEmployee" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureIncomeTaxesChangeInDeferredTaxValuationAllowanceDetails" xlink:type="extended" xlink:title="40902 - Disclosure - Income Taxes - Change in Deferred Tax Valuation Allowance (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ValuationAllowancesAndReservesTypeAxis" xlink:label="us-gaap_ValuationAllowancesAndReservesTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_ValuationAllowancesAndReservesTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ValuationAllowancesAndReservesDomain" xlink:label="us-gaap_ValuationAllowancesAndReservesDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_ValuationAllowancesAndReservesTypeAxis" xlink:to="us-gaap_ValuationAllowancesAndReservesDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ValuationAllowancesAndReservesDomain" xlink:label="us-gaap_ValuationAllowancesAndReservesDomain_636552439053564657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_ValuationAllowancesAndReservesTypeAxis" xlink:to="us-gaap_ValuationAllowancesAndReservesDomain_636552439053564657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ValuationAllowanceOfDeferredTaxAssetsMember" xlink:label="us-gaap_ValuationAllowanceOfDeferredTaxAssetsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ValuationAllowancesAndReservesDomain" xlink:to="us-gaap_ValuationAllowanceOfDeferredTaxAssetsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ValuationAllowanceAbstract" xlink:label="us-gaap_ValuationAllowanceAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ValuationAllowanceAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ValuationAllowancesAndReservesBalance" xlink:label="us-gaap_ValuationAllowancesAndReservesBalance" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ValuationAllowanceAbstract" xlink:to="us-gaap_ValuationAllowancesAndReservesBalance" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ValuationAllowancesAndReservesChargedToCostAndExpense" xlink:label="us-gaap_ValuationAllowancesAndReservesChargedToCostAndExpense" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ValuationAllowanceAbstract" xlink:to="us-gaap_ValuationAllowancesAndReservesChargedToCostAndExpense" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ValuationAllowancesAndReservesDeductions" xlink:label="us-gaap_ValuationAllowancesAndReservesDeductions" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ValuationAllowanceAbstract" xlink:to="us-gaap_ValuationAllowancesAndReservesDeductions" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureIncomeTaxesCarryforwardsDetails" xlink:type="extended" xlink:title="40903 - Disclosure - Income Taxes - Carryforwards (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_NetOperatingLossAndTaxCreditCarryOversTable" xlink:label="twou_NetOperatingLossAndTaxCreditCarryOversTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_IncomeTaxAuthorityAxis" xlink:label="us-gaap_IncomeTaxAuthorityAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="twou_NetOperatingLossAndTaxCreditCarryOversTable" xlink:to="us-gaap_IncomeTaxAuthorityAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_IncomeTaxAuthorityDomain" xlink:label="us-gaap_IncomeTaxAuthorityDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_IncomeTaxAuthorityAxis" xlink:to="us-gaap_IncomeTaxAuthorityDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_IncomeTaxAuthorityDomain" xlink:label="us-gaap_IncomeTaxAuthorityDomain_636552439053564657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_IncomeTaxAuthorityAxis" xlink:to="us-gaap_IncomeTaxAuthorityDomain_636552439053564657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_DomesticCountryMember" xlink:label="us-gaap_DomesticCountryMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_IncomeTaxAuthorityDomain" xlink:to="us-gaap_DomesticCountryMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ForeignCountryMember" xlink:label="us-gaap_ForeignCountryMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_IncomeTaxAuthorityDomain" xlink:to="us-gaap_ForeignCountryMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StatementScenarioAxis" xlink:label="us-gaap_StatementScenarioAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="twou_NetOperatingLossAndTaxCreditCarryOversTable" xlink:to="us-gaap_StatementScenarioAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScenarioUnspecifiedDomain" xlink:label="us-gaap_ScenarioUnspecifiedDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementScenarioAxis" xlink:to="us-gaap_ScenarioUnspecifiedDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScenarioUnspecifiedDomain" xlink:label="us-gaap_ScenarioUnspecifiedDomain_636552439053564657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_StatementScenarioAxis" xlink:to="us-gaap_ScenarioUnspecifiedDomain_636552439053564657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScenarioForecastMember" xlink:label="us-gaap_ScenarioForecastMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ScenarioUnspecifiedDomain" xlink:to="us-gaap_ScenarioForecastMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_StatementLineItems" xlink:to="twou_NetOperatingLossAndTaxCreditCarryOversTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_OperatingLossCarryforwards" xlink:label="us-gaap_OperatingLossCarryforwards" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLossCarryforwards" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ValuationAllowanceDeferredTaxAssetChangeInAmount" xlink:label="us-gaap_ValuationAllowanceDeferredTaxAssetChangeInAmount" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ValuationAllowanceDeferredTaxAssetChangeInAmount" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_IncomeTaxReturnsUnderExamination" xlink:label="twou_IncomeTaxReturnsUnderExamination" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_StatementLineItems" xlink:to="twou_IncomeTaxReturnsUnderExamination" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_TaxCutsAndJobsActOf2017TransitionTaxForAccumulatedForeignEarningsIncomeTaxExpense" xlink:label="twou_TaxCutsAndJobsActOf2017TransitionTaxForAccumulatedForeignEarningsIncomeTaxExpense" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_StatementLineItems" xlink:to="twou_TaxCutsAndJobsActOf2017TransitionTaxForAccumulatedForeignEarningsIncomeTaxExpense" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureStockholdersEquityDetails" xlink:type="extended" xlink:title="41001 - Disclosure - Stockholders' Equity (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable" xlink:label="us-gaap_SubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_SubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable" xlink:to="us-gaap_StatementEquityComponentsAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_CommonStockMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_SubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain_636552439053564657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain_636552439053564657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_OverAllotmentOptionMember" xlink:label="us-gaap_OverAllotmentOptionMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="us-gaap_OverAllotmentOptionMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_SubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable" xlink:to="us-gaap_PlanNameAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain_636552439053564657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain_636552439053564657" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_EquityIncentivePlan2014Member" xlink:label="twou_EquityIncentivePlan2014Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_PlanNameDomain" xlink:to="twou_EquityIncentivePlan2014Member" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SubsidiarySaleOfStockLineItems" xlink:label="us-gaap_SubsidiarySaleOfStockLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_SubsidiarySaleOfStockLineItems" xlink:to="us-gaap_SubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_SubsidiarySaleOfStockLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:label="us-gaap_ProceedsFromIssuanceOfCommonStock" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_SubsidiarySaleOfStockLineItems" xlink:to="us-gaap_ProceedsFromIssuanceOfCommonStock" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_CapitalStockSharesAuthorized" xlink:label="twou_CapitalStockSharesAuthorized" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_SubsidiarySaleOfStockLineItems" xlink:to="twou_CapitalStockSharesAuthorized" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_CommonStockSharesAuthorized" xlink:label="us-gaap_CommonStockSharesAuthorized" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_SubsidiarySaleOfStockLineItems" xlink:to="us-gaap_CommonStockSharesAuthorized" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PreferredStockSharesAuthorized" xlink:label="us-gaap_PreferredStockSharesAuthorized" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_SubsidiarySaleOfStockLineItems" xlink:to="us-gaap_PreferredStockSharesAuthorized" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_CommonStockCapitalSharesReservedForFutureIssuanceAbstract" xlink:label="twou_CommonStockCapitalSharesReservedForFutureIssuanceAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_SubsidiarySaleOfStockLineItems" xlink:to="twou_CommonStockCapitalSharesReservedForFutureIssuanceAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_CommonStockCapitalSharesReservedForFutureIssuanceOutstandingStockOptions" xlink:label="twou_CommonStockCapitalSharesReservedForFutureIssuanceOutstandingStockOptions" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="twou_CommonStockCapitalSharesReservedForFutureIssuanceAbstract" xlink:to="twou_CommonStockCapitalSharesReservedForFutureIssuanceOutstandingStockOptions" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_CommonStockCapitalSharesReservedForFutureIssuancePossibleFutureIssuanceUnderStockOptionPlans" xlink:label="twou_CommonStockCapitalSharesReservedForFutureIssuancePossibleFutureIssuanceUnderStockOptionPlans" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="twou_CommonStockCapitalSharesReservedForFutureIssuanceAbstract" xlink:to="twou_CommonStockCapitalSharesReservedForFutureIssuancePossibleFutureIssuanceUnderStockOptionPlans" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_CommonStockCapitalSharesReservedForFutureIssuanceOutstandingRestrictedStockUnits" xlink:label="twou_CommonStockCapitalSharesReservedForFutureIssuanceOutstandingRestrictedStockUnits" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="twou_CommonStockCapitalSharesReservedForFutureIssuanceAbstract" xlink:to="twou_CommonStockCapitalSharesReservedForFutureIssuanceOutstandingRestrictedStockUnits" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_CommonStockCapitalSharesReservedForFutureIssuanceAvailableForFutureIssuanceUnderStockPurchasePlan" xlink:label="twou_CommonStockCapitalSharesReservedForFutureIssuanceAvailableForFutureIssuanceUnderStockPurchasePlan" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="twou_CommonStockCapitalSharesReservedForFutureIssuanceAbstract" xlink:to="twou_CommonStockCapitalSharesReservedForFutureIssuanceAvailableForFutureIssuanceUnderStockPurchasePlan" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" xlink:label="us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="twou_CommonStockCapitalSharesReservedForFutureIssuanceAbstract" xlink:to="us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureStockBasedCompensationExpenseDetails" xlink:type="extended" xlink:title="41101 - Disclosure - Stock-Based Compensation - Expense (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="us-gaap_PlanNameAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_EquityIncentivePlan2008Member" xlink:label="twou_EquityIncentivePlan2008Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_PlanNameDomain" xlink:to="twou_EquityIncentivePlan2008Member" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_EquityIncentivePlan2014Member" xlink:label="twou_EquityIncentivePlan2014Member" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_PlanNameDomain" xlink:to="twou_EquityIncentivePlan2014Member" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="us-gaap_AwardTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_PerformanceSharesMember" xlink:label="us-gaap_PerformanceSharesMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_PerformanceSharesMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RangeAxis" xlink:label="us-gaap_RangeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="us-gaap_RangeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RangeMember" xlink:label="us-gaap_RangeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_RangeAxis" xlink:to="us-gaap_RangeMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_MaximumMember" xlink:label="us-gaap_MaximumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_RangeMember" xlink:to="us-gaap_MaximumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_IncomeStatementLocationAxis" xlink:label="us-gaap_IncomeStatementLocationAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="4" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="us-gaap_IncomeStatementLocationAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="us-gaap_IncomeStatementLocationDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="us-gaap_IncomeStatementLocationDomain_636552439053564657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain_636552439053564657" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_CurriculumAndTeachingMember" xlink:label="twou_CurriculumAndTeachingMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="twou_CurriculumAndTeachingMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_ServicingAndSupportMember" xlink:label="twou_ServicingAndSupportMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="twou_ServicingAndSupportMember" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_TechnologyAndContentDevelopmentMember" xlink:label="twou_TechnologyAndContentDevelopmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="twou_TechnologyAndContentDevelopmentMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SellingAndMarketingExpenseMember" xlink:label="us-gaap_SellingAndMarketingExpenseMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_SellingAndMarketingExpenseMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_GeneralAndAdministrativeExpenseMember" xlink:label="us-gaap_GeneralAndAdministrativeExpenseMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_GeneralAndAdministrativeExpenseMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_ShareBasedCompensationArrangementsByShareBasedPaymentAwardNumberOfEmployeeCompensationPlans" xlink:label="twou_ShareBasedCompensationArrangementsByShareBasedPaymentAwardNumberOfEmployeeCompensationPlans" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="twou_ShareBasedCompensationArrangementsByShareBasedPaymentAwardNumberOfEmployeeCompensationPlans" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAuthorized" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAuthorized" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAuthorized" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfAdditionalSharesAuthorized" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfAdditionalSharesAuthorized" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfAdditionalSharesAuthorized" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_ShareBasedCompensationArrangementByShareBasedPaymentAwardPeriodOfAnnualAutomaticIncreaseInSharesAuthorized" xlink:label="twou_ShareBasedCompensationArrangementByShareBasedPaymentAwardPeriodOfAnnualAutomaticIncreaseInSharesAuthorized" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="twou_ShareBasedCompensationArrangementByShareBasedPaymentAwardPeriodOfAnnualAutomaticIncreaseInSharesAuthorized" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_ShareBasedCompensationArrangementByShareBasedPaymentAwardPercentageAppliedOnOutstandingSharesOfCommonStockForAutomaticInclusionInPlan" xlink:label="twou_ShareBasedCompensationArrangementByShareBasedPaymentAwardPercentageAppliedOnOutstandingSharesOfCommonStockForAutomaticInclusionInPlan" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="twou_ShareBasedCompensationArrangementByShareBasedPaymentAwardPercentageAppliedOnOutstandingSharesOfCommonStockForAutomaticInclusionInPlan" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" xlink:label="us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="12" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="13" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGranted" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGranted" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="14" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGranted" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="15" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureStockBasedCompensationOtherDetails" xlink:type="extended" xlink:title="41102 - Disclosure - Stock-Based Compensation - Other (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RangeAxis" xlink:label="us-gaap_RangeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_RangeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RangeMember" xlink:label="us-gaap_RangeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_RangeAxis" xlink:to="us-gaap_RangeMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_MinimumMember" xlink:label="us-gaap_MinimumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_RangeMember" xlink:to="us-gaap_MinimumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_MaximumMember" xlink:label="us-gaap_MaximumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_RangeMember" xlink:to="us-gaap_MaximumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMinimum" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMinimum" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMinimum" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMaximum" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMaximum" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMaximum" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMinimum" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMinimum" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMinimum" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMaximum" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMaximum" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMaximum" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsAndMethodologyAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExpirationsInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExpirationsInPeriod" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExpirationsInPeriod" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableNumber" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableNumber" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingNumber" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingNumber" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExpirationsInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExpirationsInPeriodWeightedAverageExercisePrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExpirationsInPeriodWeightedAverageExercisePrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableWeightedAverageExercisePrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableWeightedAverageExercisePrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageExercisePrice" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageExercisePrice" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsWeightedAverageRemainingContractualTermAbstract" xlink:label="twou_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsWeightedAverageRemainingContractualTermAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="twou_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsWeightedAverageRemainingContractualTermAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="twou_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsWeightedAverageRemainingContractualTermAbstract" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsGrantsInPeriodWeightedAverageRemainingContractualTerm" xlink:label="twou_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsGrantsInPeriodWeightedAverageRemainingContractualTerm" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="twou_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsWeightedAverageRemainingContractualTermAbstract" xlink:to="twou_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsGrantsInPeriodWeightedAverageRemainingContractualTerm" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisesInPeriodWeightedAverageRemainingContractualTerm" xlink:label="twou_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisesInPeriodWeightedAverageRemainingContractualTerm" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="twou_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsWeightedAverageRemainingContractualTermAbstract" xlink:to="twou_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisesInPeriodWeightedAverageRemainingContractualTerm" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableWeightedAverageRemainingContractualTerm1" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableWeightedAverageRemainingContractualTerm1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="twou_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsWeightedAverageRemainingContractualTermAbstract" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableWeightedAverageRemainingContractualTerm1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageRemainingContractualTerm1" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageRemainingContractualTerm1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="twou_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsWeightedAverageRemainingContractualTermAbstract" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingWeightedAverageRemainingContractualTerm1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsIntrinsicValueAbstract" xlink:label="twou_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsIntrinsicValueAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="twou_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsIntrinsicValueAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="twou_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsIntrinsicValueAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableIntrinsicValue1" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableIntrinsicValue1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="twou_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsIntrinsicValueAbstract" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableIntrinsicValue1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingAggregateIntrinsicValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingAggregateIntrinsicValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="twou_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsIntrinsicValueAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestOutstandingAggregateIntrinsicValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingRollForward" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingRollForward" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingRollForward" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGranted" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGranted" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGranted" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingRollForward" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedMaximumPeriodForRecognition1" xlink:label="twou_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedMaximumPeriodForRecognition1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingRollForward" xlink:to="twou_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedMaximumPeriodForRecognition1" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsAdditionalDisclosuresAbstract" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsAdditionalDisclosuresAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsAdditionalDisclosuresAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsAdditionalDisclosuresAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsAdditionalDisclosuresAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsAdditionalDisclosuresAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsAdditionalDisclosuresAbstract" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureStockBasedCompensationEmployeeStockPurchasePlanDetails" xlink:type="extended" xlink:title="41103 - Disclosure - Stock-Based Compensation - Employee Stock Purchase Plan (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RangeAxis" xlink:label="us-gaap_RangeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_RangeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RangeMember" xlink:label="us-gaap_RangeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_RangeAxis" xlink:to="us-gaap_RangeMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_MinimumMember" xlink:label="us-gaap_MinimumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_RangeMember" xlink:to="us-gaap_MinimumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_MaximumMember" xlink:label="us-gaap_MaximumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_RangeMember" xlink:to="us-gaap_MaximumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_EmployeeStockPurchasePlanAbstract" xlink:label="twou_EmployeeStockPurchasePlanAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="twou_EmployeeStockPurchasePlanAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_PercentagePurchasePriceFairMarketValue" xlink:label="twou_PercentagePurchasePriceFairMarketValue" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="twou_EmployeeStockPurchasePlanAbstract" xlink:to="twou_PercentagePurchasePriceFairMarketValue" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_DefinedContributionPlanAnnualContributionsPerEmployeePercent" xlink:label="twou_DefinedContributionPlanAnnualContributionsPerEmployeePercent" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="twou_EmployeeStockPurchasePlanAbstract" xlink:to="twou_DefinedContributionPlanAnnualContributionsPerEmployeePercent" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_DefinedContributionPlanMaximumAnnualContributionsPerEmployeeAmount" xlink:label="us-gaap_DefinedContributionPlanMaximumAnnualContributionsPerEmployeeAmount" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="twou_EmployeeStockPurchasePlanAbstract" xlink:to="us-gaap_DefinedContributionPlanMaximumAnnualContributionsPerEmployeeAmount" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:label="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="twou_EmployeeStockPurchasePlanAbstract" xlink:to="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureNetLossPerShareAntidilutiveDetails" xlink:type="extended" xlink:title="41201 - Disclosure - Net Loss per Share - Antidilutive (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:label="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain_636552439053720657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain_636552439053720657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:to="us-gaap_AwardTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:to="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureSegmentAndGeographicInformationConcentrationRiskDetails" xlink:type="extended" xlink:title="41301 - Disclosure - Segment and Geographic Information - Concentration Risk (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:label="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ConcentrationRiskByBenchmarkAxis" xlink:label="us-gaap_ConcentrationRiskByBenchmarkAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="us-gaap_ConcentrationRiskByBenchmarkAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ConcentrationRiskBenchmarkDomain" xlink:label="us-gaap_ConcentrationRiskBenchmarkDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_ConcentrationRiskByBenchmarkAxis" xlink:to="us-gaap_ConcentrationRiskBenchmarkDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ConcentrationRiskBenchmarkDomain" xlink:label="us-gaap_ConcentrationRiskBenchmarkDomain_636552439053720657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_ConcentrationRiskByBenchmarkAxis" xlink:to="us-gaap_ConcentrationRiskBenchmarkDomain_636552439053720657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SalesRevenueServicesNetMember" xlink:label="us-gaap_SalesRevenueServicesNetMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ConcentrationRiskBenchmarkDomain" xlink:to="us-gaap_SalesRevenueServicesNetMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AccountsReceivableMember" xlink:label="us-gaap_AccountsReceivableMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ConcentrationRiskBenchmarkDomain" xlink:to="us-gaap_AccountsReceivableMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ConcentrationRiskByTypeAxis" xlink:label="us-gaap_ConcentrationRiskByTypeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="us-gaap_ConcentrationRiskByTypeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ConcentrationRiskTypeDomain" xlink:label="us-gaap_ConcentrationRiskTypeDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_ConcentrationRiskByTypeAxis" xlink:to="us-gaap_ConcentrationRiskTypeDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ConcentrationRiskTypeDomain" xlink:label="us-gaap_ConcentrationRiskTypeDomain_636552439053720657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_ConcentrationRiskByTypeAxis" xlink:to="us-gaap_ConcentrationRiskTypeDomain_636552439053720657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_CustomerConcentrationRiskMember" xlink:label="us-gaap_CustomerConcentrationRiskMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ConcentrationRiskTypeDomain" xlink:to="us-gaap_CustomerConcentrationRiskMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_CreditConcentrationRiskMember" xlink:label="us-gaap_CreditConcentrationRiskMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ConcentrationRiskTypeDomain" xlink:to="us-gaap_CreditConcentrationRiskMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_MajorCustomersAxis" xlink:label="us-gaap_MajorCustomersAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="us-gaap_MajorCustomersAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_NameOfMajorCustomerDomain" xlink:label="us-gaap_NameOfMajorCustomerDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_MajorCustomersAxis" xlink:to="us-gaap_NameOfMajorCustomerDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_NameOfMajorCustomerDomain" xlink:label="us-gaap_NameOfMajorCustomerDomain_636552439053720657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_MajorCustomersAxis" xlink:to="us-gaap_NameOfMajorCustomerDomain_636552439053720657" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_UniversityClientOneMember" xlink:label="twou_UniversityClientOneMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_NameOfMajorCustomerDomain" xlink:to="twou_UniversityClientOneMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_UniversityClientTwoMember" xlink:label="twou_UniversityClientTwoMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_NameOfMajorCustomerDomain" xlink:to="twou_UniversityClientTwoMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_UniversityClientThreeMember" xlink:label="twou_UniversityClientThreeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_NameOfMajorCustomerDomain" xlink:to="twou_UniversityClientThreeMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_UniversityClientFourMember" xlink:label="twou_UniversityClientFourMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_NameOfMajorCustomerDomain" xlink:to="twou_UniversityClientFourMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StatementBusinessSegmentsAxis" xlink:label="us-gaap_StatementBusinessSegmentsAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="4" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="us-gaap_StatementBusinessSegmentsAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SegmentDomain" xlink:label="us-gaap_SegmentDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementBusinessSegmentsAxis" xlink:to="us-gaap_SegmentDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_GraduateProgramSegmentMember" xlink:label="twou_GraduateProgramSegmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SegmentDomain" xlink:to="twou_GraduateProgramSegmentMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_ShortCourseSegmentMember" xlink:label="twou_ShortCourseSegmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_SegmentDomain" xlink:to="twou_ShortCourseSegmentMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SegmentReportingInformationLineItems" xlink:label="us-gaap_SegmentReportingInformationLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_NumberOfOperatingSegments" xlink:label="us-gaap_NumberOfOperatingSegments" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_NumberOfOperatingSegments" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_NumberOfReportableSegments" xlink:label="us-gaap_NumberOfReportableSegments" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_NumberOfReportableSegments" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_NumberOfClients" xlink:label="twou_NumberOfClients" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="twou_NumberOfClients" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SalesRevenueServicesNet" xlink:label="us-gaap_SalesRevenueServicesNet" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_SalesRevenueServicesNet" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_AccountsReceivableNetCurrent" xlink:label="us-gaap_AccountsReceivableNetCurrent" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_AccountsReceivableNetCurrent" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ConcentrationRiskPercentage1" xlink:label="us-gaap_ConcentrationRiskPercentage1" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_ConcentrationRiskPercentage1" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureSegmentAndGeographicInformationSegmentPerformanceDetails" xlink:type="extended" xlink:title="41302 - Disclosure - Segment and Geographic Information - Segment Performance (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:label="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StatementBusinessSegmentsAxis" xlink:label="us-gaap_StatementBusinessSegmentsAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="us-gaap_StatementBusinessSegmentsAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SegmentDomain" xlink:label="us-gaap_SegmentDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementBusinessSegmentsAxis" xlink:to="us-gaap_SegmentDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_GraduateProgramSegmentMember" xlink:label="twou_GraduateProgramSegmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SegmentDomain" xlink:to="twou_GraduateProgramSegmentMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_ShortCourseSegmentMember" xlink:label="twou_ShortCourseSegmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_SegmentDomain" xlink:to="twou_ShortCourseSegmentMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SegmentReportingInformationLineItems" xlink:label="us-gaap_SegmentReportingInformationLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SegmentReportingInformationRevenueAbstract" xlink:label="us-gaap_SegmentReportingInformationRevenueAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_SegmentReportingInformationRevenueAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SalesRevenueServicesNet" xlink:label="us-gaap_SalesRevenueServicesNet" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SegmentReportingInformationRevenueAbstract" xlink:to="us-gaap_SalesRevenueServicesNet" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SegmentReportingInformationProfitLossAbstract" xlink:label="us-gaap_SegmentReportingInformationProfitLossAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_SegmentReportingInformationProfitLossAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_ProfitLossFromSegmentOperations" xlink:label="twou_ProfitLossFromSegmentOperations" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SegmentReportingInformationProfitLossAbstract" xlink:to="twou_ProfitLossFromSegmentOperations" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_SegmentProfitabilityMarginAbstract" xlink:label="twou_SegmentProfitabilityMarginAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="twou_SegmentProfitabilityMarginAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_PercentageOfProfitLossFromSegmentOperations" xlink:label="twou_PercentageOfProfitLossFromSegmentOperations" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="twou_SegmentProfitabilityMarginAbstract" xlink:to="twou_PercentageOfProfitLossFromSegmentOperations" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_NetIncomeLossAbstract" xlink:label="us-gaap_NetIncomeLossAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_NetIncomeLossAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_NetIncomeLossAbstract" xlink:to="us-gaap_NetIncomeLoss" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_SegmentReportingInformationAdjustmentsToNetIncomeLossAbstract" xlink:label="twou_SegmentReportingInformationAdjustmentsToNetIncomeLossAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_NetIncomeLossAbstract" xlink:to="twou_SegmentReportingInformationAdjustmentsToNetIncomeLossAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_InvestmentIncomeInterest" xlink:label="us-gaap_InvestmentIncomeInterest" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="twou_SegmentReportingInformationAdjustmentsToNetIncomeLossAbstract" xlink:to="us-gaap_InvestmentIncomeInterest" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_InterestExpense" xlink:label="us-gaap_InterestExpense" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="twou_SegmentReportingInformationAdjustmentsToNetIncomeLossAbstract" xlink:to="us-gaap_InterestExpense" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ForeignCurrencyTransactionLossBeforeTax" xlink:label="us-gaap_ForeignCurrencyTransactionLossBeforeTax" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="twou_SegmentReportingInformationAdjustmentsToNetIncomeLossAbstract" xlink:to="us-gaap_ForeignCurrencyTransactionLossBeforeTax" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_DepreciationDepletionAndAmortization" xlink:label="us-gaap_DepreciationDepletionAndAmortization" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="twou_SegmentReportingInformationAdjustmentsToNetIncomeLossAbstract" xlink:to="us-gaap_DepreciationDepletionAndAmortization" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_IncomeTaxExpenseBenefit" xlink:label="us-gaap_IncomeTaxExpenseBenefit" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="twou_SegmentReportingInformationAdjustmentsToNetIncomeLossAbstract" xlink:to="us-gaap_IncomeTaxExpenseBenefit" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ShareBasedCompensation" xlink:label="us-gaap_ShareBasedCompensation" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="twou_SegmentReportingInformationAdjustmentsToNetIncomeLossAbstract" xlink:to="us-gaap_ShareBasedCompensation" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_AdjustmentsToNetIncomeLoss" xlink:label="twou_AdjustmentsToNetIncomeLoss" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="twou_SegmentReportingInformationAdjustmentsToNetIncomeLossAbstract" xlink:to="twou_AdjustmentsToNetIncomeLoss" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_SegmentReportingInformationAssetsAbstract" xlink:label="twou_SegmentReportingInformationAssetsAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="twou_SegmentReportingInformationAssetsAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_Assets" xlink:label="us-gaap_Assets" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="twou_SegmentReportingInformationAssetsAbstract" xlink:to="us-gaap_Assets" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureSegmentAndGeographicInformationGeographicalInformationDetails" xlink:type="extended" xlink:title="41303 - Disclosure - Segment and Geographic Information - Geographical Information (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:label="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StatementBusinessSegmentsAxis" xlink:label="us-gaap_StatementBusinessSegmentsAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="us-gaap_StatementBusinessSegmentsAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SegmentDomain" xlink:label="us-gaap_SegmentDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementBusinessSegmentsAxis" xlink:to="us-gaap_SegmentDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_ShortCourseSegmentMember" xlink:label="twou_ShortCourseSegmentMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SegmentDomain" xlink:to="twou_ShortCourseSegmentMember" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_StatementGeographicalAxis" xlink:label="us-gaap_StatementGeographicalAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="us-gaap_StatementGeographicalAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SegmentGeographicalDomain" xlink:label="us-gaap_SegmentGeographicalDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementGeographicalAxis" xlink:to="us-gaap_SegmentGeographicalDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SegmentGeographicalDomain" xlink:label="us-gaap_SegmentGeographicalDomain_636552439053720657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_StatementGeographicalAxis" xlink:to="us-gaap_SegmentGeographicalDomain_636552439053720657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_NonUsMember" xlink:label="us-gaap_NonUsMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SegmentGeographicalDomain" xlink:to="us-gaap_NonUsMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SegmentReportingInformationLineItems" xlink:label="us-gaap_SegmentReportingInformationLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SegmentsGeographicalAreasAbstract" xlink:label="us-gaap_SegmentsGeographicalAreasAbstract" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_SegmentsGeographicalAreasAbstract" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_SalesRevenueServicesNet" xlink:label="us-gaap_SalesRevenueServicesNet" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SegmentsGeographicalAreasAbstract" xlink:to="us-gaap_SalesRevenueServicesNet" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_NoncurrentAssets" xlink:label="us-gaap_NoncurrentAssets" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_SegmentsGeographicalAreasAbstract" xlink:to="us-gaap_NoncurrentAssets" priority="1" use="optional" />
  </definitionLink>
  <definitionLink xlink:role="http://www.2u.com/role/DisclosureRelatedPartyTransactionsDetails" xlink:type="extended" xlink:title="41501 - Disclosure - Related Party Transactions (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable" xlink:label="us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RangeAxis" xlink:label="us-gaap_RangeAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable" xlink:to="us-gaap_RangeAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RangeMember" xlink:label="us-gaap_RangeMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_RangeAxis" xlink:to="us-gaap_RangeMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_MinimumMember" xlink:label="us-gaap_MinimumMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_RangeMember" xlink:to="us-gaap_MinimumMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:label="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable" xlink:to="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:to="us-gaap_RelatedPartyDomain" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain_636552439053720657" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:to="us-gaap_RelatedPartyDomain_636552439053720657" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_ExecutiveOfficerMember" xlink:label="us-gaap_ExecutiveOfficerMember" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_RelatedPartyDomain" xlink:to="us-gaap_ExecutiveOfficerMember" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RelatedPartyTransactionLineItems" xlink:label="us-gaap_RelatedPartyTransactionLineItems" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_RelatedPartyTransactionLineItems" xlink:to="us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_OwnershipInterest" xlink:label="twou_OwnershipInterest" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_RelatedPartyTransactionLineItems" xlink:to="twou_OwnershipInterest" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_RepaymentOfSecurityDepositFromRelatedParty" xlink:label="twou_RepaymentOfSecurityDepositFromRelatedParty" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_RelatedPartyTransactionLineItems" xlink:to="twou_RepaymentOfSecurityDepositFromRelatedParty" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="twou-20171231.xsd#twou_RentalIncomeFromRelatedParties" xlink:label="twou_RentalIncomeFromRelatedParties" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_RelatedPartyTransactionLineItems" xlink:to="twou_RentalIncomeFromRelatedParties" priority="1" use="optional" />
    <loc xlink:type="locator" xlink:href="http://xbrl.fasb.org/us-gaap/2017/elts/us-gaap-2017-01-31.xsd#us-gaap_RelatedPartyTransactionExpensesFromTransactionsWithRelatedParty" xlink:label="us-gaap_RelatedPartyTransactionExpensesFromTransactionsWithRelatedParty" />
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_RelatedPartyTransactionLineItems" xlink:to="us-gaap_RelatedPartyTransactionExpensesFromTransactionsWithRelatedParty" priority="1" use="optional" />
  </definitionLink>
</link:linkbase>
</XBRL>
