<?xml version="1.0"?>
<ownershipDocument>

    <schemaVersion>X0508</schemaVersion>

    <documentType>4</documentType>

    <periodOfReport>2026-03-16</periodOfReport>

    <issuer>
        <issuerCik>0002022626</issuerCik>
        <issuerName>Upstream Bio, Inc.</issuerName>
        <issuerTradingSymbol>UPB</issuerTradingSymbol>
    </issuer>

    <reportingOwner>
        <reportingOwnerId>
            <rptOwnerCik>0001271411</rptOwnerCik>
            <rptOwnerName>GRAY MICHAEL</rptOwnerName>
        </reportingOwnerId>
        <reportingOwnerAddress>
            <rptOwnerStreet1>UPSTREAM BIO, INC.</rptOwnerStreet1>
            <rptOwnerStreet2>890 WINTER STREET, SUITE 200</rptOwnerStreet2>
            <rptOwnerCity>WALTHAM</rptOwnerCity>
            <rptOwnerState>MA</rptOwnerState>
            <rptOwnerZipCode>02451</rptOwnerZipCode>
            <rptOwnerStateDescription></rptOwnerStateDescription>
        </reportingOwnerAddress>
        <reportingOwnerRelationship>
            <isDirector>false</isDirector>
            <isOfficer>true</isOfficer>
            <isTenPercentOwner>false</isTenPercentOwner>
            <isOther>false</isOther>
            <officerTitle>CFO and COO</officerTitle>
        </reportingOwnerRelationship>
    </reportingOwner>

    <aff10b5One>false</aff10b5One>

    <nonDerivativeTable>
        <nonDerivativeTransaction>
            <securityTitle>
                <value>Common Stock</value>
            </securityTitle>
            <transactionDate>
                <value>2026-03-16</value>
            </transactionDate>
            <transactionCoding>
                <transactionFormType>4</transactionFormType>
                <transactionCode>S</transactionCode>
                <equitySwapInvolved>false</equitySwapInvolved>
                <footnoteId id="F1"/>
            </transactionCoding>
            <transactionAmounts>
                <transactionShares>
                    <value>852</value>
                </transactionShares>
                <transactionPricePerShare>
                    <value>9.29</value>
                </transactionPricePerShare>
                <transactionAcquiredDisposedCode>
                    <value>D</value>
                </transactionAcquiredDisposedCode>
            </transactionAmounts>
            <postTransactionAmounts>
                <sharesOwnedFollowingTransaction>
                    <value>29648</value>
                </sharesOwnedFollowingTransaction>
            </postTransactionAmounts>
            <ownershipNature>
                <directOrIndirectOwnership>
                    <value>D</value>
                </directOrIndirectOwnership>
            </ownershipNature>
        </nonDerivativeTransaction>
    </nonDerivativeTable>

    <footnotes>
        <footnote id="F1">The Issuer has adopted a &quot;sell-to-cover&quot; policy to satisfy the tax withholding obligations of the Reporting Person. The sales reported on this Form 4 represent the number of shares required to be sold by the Reporting Person to cover tax withholding obligations in connection with the vesting of restricted stock units. Such sales were automatic and not at the discretion of the Reporting Person.</footnote>
    </footnotes>

    <ownerSignature>
        <signatureName>/s/ Allison Ambrose, Attorney-in-Fact</signatureName>
        <signatureDate>2026-03-17</signatureDate>
    </ownerSignature>
</ownershipDocument>
