The study of human thymopoiesis has been hampered by our inability to provide all the signals of the normal thymic microenvironment to human stem and progenitor cells in vitro. Although T cell production from CD34+ progenitors can be achieved in vitro, purified populations of the immature CD34+CD38- cells (which include HSC) do not generate T cells reliably with the existing assays. The most commonly cited system for human T cell assay, Fetal thymic organ culture (FTOC) from immune deficient mice, is logistically very difficult and low seeding efficiency precludes its use for clonal assays. Recently, a murine bone marrow stromal line engineered to constitutively express Delta-1 ligand (OP9-DL1 stroma), has been shown to support production of mature murine T cells from HSC [Schmitt, 2002] and ES cells [Schmitt, 2004]. Here we report the use of modifications to the OP9-DL1 system that allow assay of human T lymphopoiesis from HSC and progenitors. CD34+lin-CD38- cells (HSC) from cord blood (CB) and bone marrow (BM)cultured on OP9 control stroma (that does not express DL) produced CD19+ B cells and CD56+ NK cells and maintained CD34+ cells for several weeks. However, T cell commitment was not evident in these control cultures. In contrast, culture of HSC on OP9-DL1 stroma (Dr. Zuniga-Pflucker) resulted in robust growth of T cell precursors (as shown by expression of CD7 and cytoplasmic (cy) CD3 in over 50% of cells) and mRNA for pre-Tα. NK cells were also increased on OP9-DL1 relative to control OP9 stroma but B cell production was lost. Although T cell commitment to the CD34+CD7+ and CD34-cyCD3+CD7+ cells stages was accomplished on OP9-DL1 stroma, markers of further T cell maturation such as surface CD3, CD4 and CD8, were absent on the cells produced. Multiple combinations of growth factors (IL-7, IL-2, IL-15, IL-3, ckit ligand (KL) and thrombopoietin (Tpo)) to the OP9-DL1 stromal cultures failed to achieve further T cell maturation, although the combination of KL, Tpo, IL-7 produced optimal cell growth. However, when conditioned medium collected from human thymic stromal cultures (thyCM) (medium collected from adherent cells derived from human postnatal thymus and filtered through 0.45u) was added to OP9-DL1 co-cultures, cyCD3+ cells were increased and differentiation of CD3+CD4+ cells was achieved. Thymic CD34+lin-CD7+ cells produced large numbers of CD3+CD4+CD8+, CD4+CD8- and CD4-CD8+ cells in this system. We have now used the OP9-DL1/thyCM system to demonstrate the T cell potential of CB and BM CD34+CD38-lin-CD7- (HSC) and CB CD34+CD38-lin-CD7+ (CLP). In addition, efficient cloning of single CB HSC and primitive thymic progenitors has been achieved using this culture system. The data suggests that, although DL-1 is required and sufficient for early T lymphoid commitment, other factor(s) specific to human thymic stroma are required for production of more mature T cells; these factors can now be identified using the OP9-DL1 system. The availability of a simple monolayer culture system that allows T lymphoid commitment from HSC and progenitors and further differentiation to mature T cells will be of great value in the study of the lineage potential of progenitor populations and the regulation of human thymopoiesis.