A feedback amplifier circuit with Notch and E2A orchestrates T-cell fate and suppresses the innate lymphoid cell lineages during thymic ontogeny.

External signals from the thymic microenvironment and the activities of lineage-specific transcription factors (TFs) instruct T-cell versus innate lymphoid cell (ILC) fates. However, mechanistic insights into how factors such as Notch1-Delta-like-4 (Dll4) signaling and E-protein TFs collaborate to establish T-cell identity remain rudimentary. Using multiple in vivo approaches and single-cell multiome analysis, we identified a feedback amplifier circuit that specifies fetal and adult T-cell fates. In early T progenitors (ETPs) in the fetal thymus, Notch signaling minimally lowered E-protein antagonist levels, and high abundance favored the differentiation of ETPs into ILCs. Conversely, in the adult thymus, Notch signaling markedly decreased abundance in ETPs, substantially elevating E-protein DNA binding and in turn promoting the activation of a T-cell lineage-specific gene expression program linked with V(D)J gene recombination and T-cell receptor signaling. Our findings indicate that, in the fetal versus the adult thymus, a simple feedback amplifier circuit dictated by Notch-mediated signals and abundance enforces T-cell identity and suppresses ILC development.