The identity and lineage potential of the cells that initiate thymopoiesis remain controversial. The goal of these studies was to determine, at a clonal level, the immunophenotype and differentiation pathways of the earliest progenitors in human thymus. Although the majority of human CD34(+)lin(-) thymocytes express high levels of CD7, closer analysis reveals that a continuum of CD7 expression exists, and 1% to 2% of progenitors are CD7(-). CD34(+)lin(-) thymocytes were fractionated by CD7 expression and tested for lineage potential in B-lymphoid, T-lymphoid, and myeloid-erythroid conditions. Progressive restriction in lineage potential correlated with CD7 expression, that is, the CD7(hi) fraction produced T and NK cells but lacked B and myelo-erythroid potential, the CD7(int) (CD10(+)) fraction produced B, T, and NK cells, but lacked myelo-erythroid potential. The CD7(-) fraction produced all lymphoid and myelo-erythroid lineages and expressed HSC-associated genes. However, CD34(+)lin(-)CD7(-) thymocytes also expressed early T lymphoid genes Tdt, pTalpha, and IL-7Ralpha and lacked engraftment capacity, suggesting the signals that direct lymphoid commitment and corresponding loss of HSC function are rapidly initiated on arrival of HSC in the human thymus. Thus, differential levels of CD7 identify the progressive stages of lineage commitment in human thymus, initiated from a primitive CD7(-) lympho-myeloid thymic progenitor.