It is a longstanding question which bone marrow–derived cell seeds the thymus and to what level this cell is committed to the T-cell lineage. We sought to elucidate this issue by examining gene expression, lineage potential, and self-renewal capacity of the 2 most immature subsets in the human thymus, namely CD34⁺CD1a^– and CD34⁺CD1a⁺ thymocytes. DNA microarrays revealed the presence of several myeloid and erythroid transcripts in CD34⁺CD1a^– thymocytes but not in CD34⁺CD1a⁺ thymocytes. Lineage potential of both subpopulations was assessed using in vitro colony assays, bone marrow stroma cultures, and in vivo transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. The CD34⁺CD1a^– subset contained progenitors with lymphoid (both T and B), myeloid, and erythroid lineage potential. Remarkably, development of CD34⁺CD1a^– thymocytes toward the T-cell lineage, as shown by T-cell receptor δ gene rearrangements, could be reversed into a myeloid-cell fate. In contrast, the CD34⁺CD1a⁺ cells yielded only T-cell progenitors, demonstrating their irreversible commitment to the T-cell lineage. Both CD34⁺CD1a^– and CD34⁺CD1a⁺ thymocytes failed to repopulate NOD/SCID mice. We conclude that the human thymus is seeded by multipotent progenitors with a much broader lineage potential than previously assumed. These cells resemble hematopoietic stem cells but, by analogy with murine thymocytes, apparently lack sufficient self-renewal capacity.