T-cell development follows a defined set of stage-specific differentiation steps. However, molecular and cellular events occurring at early stages of human T-cell development remain to be fully elucidated. To address this, human umbilical cord blood (UCB) hematopoietic stem cells (HSCs) were induced to differentiate to the T lineage in OP9-DL1 cocultures. A developmental program involving a sequential and temporally discrete expression of key differentiation markers was revealed. Quantitative clonal analyses demonstrated that CD34⁺CD38⁻ and CD34⁺CD38^lo subsets of UCB contain a similarly high T-lineage progenitor frequency, whereas the frequency in CD34⁺CD38^+/hi cells was 5-fold lower. Delta-like/Notch-induced signals increased the T-cell progenitor frequency of CD34⁺CD38^−/lo cells differentiated on OP9-DL1, and 2 distinct progenitor subsets, CD34⁺CD45RA⁺CD7⁺⁺CD5⁻CD1a⁻ (proT1) and CD34⁺CD45RA⁺CD7⁺⁺CD5⁺CD1a⁻ (proT2), were identified and their thymus engrafting capacity was examined, with proT2 cells showing a 3-fold enhanced reconstituting capacity compared with the proT1 subset. Furthermore, in vitro–generated CD34⁺CD7⁺⁺ progenitors effectively engrafted the thymus of immunodeficient mice, which was enhanced by the addition of an IL-7/IL-7 antibody complex. Taken together, the identification of T-progenitor subsets readily generated in vitro may offer important avenues to improve cellular-based immune-reconstitution approaches.