The thymus is the major site for T cell development, particularly in children, in whom it is normally more active than in adults. We describe here an in vitro system of T cell maturation. Human bone marrow CD34+ stem cells, obtained by column purification from healthy adult donors, were cultured with or without heterologous thymic epithelial cell monolayers prepared from normal thymus of infants, removed at cardiac surgery. The culture media contained combinations of different cytokines. The pattern of T cell differentiation was assessed by immunophenotyping non adherent cells at different times in culture by flow cytometry. In the absence of thymic epithelial stroma, but with IL-2 and IL-12, the cells acquired the surface expression of CD2 and CD5, but not of CD3, CD4 and CD8. The addition of IL-7 resulted in driving cell differentiation further to a small degree of expression of CD3 and CD4 (less than 10%) after three weeks in culture. When the stem cells were cultured on a thymic epithelial cell monolayer, we observed greater cell viability and a high degree of expression of CD3 (60%), CD4 (40%) and CD8(15%) at 3 weeks of culture, which was not significantly different with either cytokine combination. However, a higher proportion of double positive CD4+CD8+ cells appeared with the IL-2 and IL-12 cytokine combination. These data support the importance of thymic stromal factors in defining growth and maturation of human stem cells. Delineating the complex factors within the thymic microenvironment that regulate T cell maturation will be of great importance in the development of therapeutic approaches for immunodeficient states requiring T cell reconstitution, such as AIDS.