We have identified a large population of CD3−7+ cells in human fetal gut. Three- and four-color flow cytometry revealed a distinct surface Ag profile on this population; the majority were negative for CD4 and CD8, whereas most of the remainder expressed the CD8αα homodimer. In contrast about half of CD3+ cells expressed CD4 and half expressed CD8α. A large proportion of CD3−7+ cells expressed CD56, CD94, and CD161, and whereas CD3+ T cells also expressed CD161, they only rarely expressed CD56 or CD94. Further studies were conducted to determine whether the CD3−7+ cells have the potential to differentiate into CD3+ cells. About half of CD3−7+ cells contain intracellular CD3ε. Rearranged TCR γ-chains were detected in highly purified CD3−7+ cells as an early molecular sign of T cell commitment, and the pattern of rearrangement with V regions spliced to the most 5′ Jγ segment is reminiscent of early thymocyte differentiation. In reaggregate thymic organ cultures, CD3−7+ cells also gave rise to CD3+ T cells. Thus, we demonstrate that the CD3−7+ cells present in the human fetal gut display a distinct phenotype and are able to develop into CD3+ T cells.