Signaling by the transmembrane receptor Notch is critical for T lineage development, but progenitor subsets that first receive Notch signals have not been defined. Here we identify an immature subset of early T lineage progenitors (ETPs) in the thymus that expressed the tyrosine kinase receptor Flt3 and had preserved B lineage potential at low progenitor frequency. Notch signaling was active in ETPs and was required for generation of the ETP population. Additionally, Notch signals contributed to the subsequent differentiation of ETPs. In contrast, multipotent hematopoietic progenitors circulated in the blood even in the absence of Notch signaling, suggesting that critical Notch signals during early T lineage development are delivered early after thymic entry.