Pluripotent lymphohematopoietic stem cells are probably confined to bone marrow cells expressing CD34 surface molecules. To investigate the capacity of adult human CD34+ bone marrow cells to differentiate along the T lymphoid lineage, we plated purified CD34+ cells from healthy adults in liquid culture on adherent thymic stromal cells prepared from HLA- or blood group-mismatched postnatal thymic tissue. We show that purified CD34+CD3-CD4-CD8- bone marrow cells contained progenitors with the ability to differentiate into CD4+ and CD8+ T lymphocytes expressing surface (s)CD3 and T cell receptor alpha/beta in vitro. These progenitors were found in the CD34+CD2+sCD3-CD4-CD8-, CD34+CD7+sCD3-CD4-CD8-, and CD34+CD2+CD7+sCD3-CD4-CD8-, as well as in the CD34+CD2-sCD3-CD4-CD8-, CD34+CD7-sCD3-CD4-CD8-, and CD34+CD2-CD7-sCD3-CD4-CD8- subsets, indicating that T lymphocyte progenitors sensitive to signals mediated by thymic stroma in vitro are not restricted to CD34+ cells already coexpressing early T lymphocyte-associated markers. Finally, we show that T lymphopoiesis was enhanced by c-kit ligand.