Introduction: Umbilical cord blood contains relatively abundant primitive CD34 + hematopoietic progenitor cells which can differentiate into T lymphocytes ex vivo. Materials and Methods: In this study, thymic stromal cells (TSCs) were isolated from aborted fetuses and a monolayer culture system was established. Highly-purified CD34 + cells from umbilical cord blood were cultured on the TSCs after limiting-dilution. The cells were then harvested and evaluated for CD4, CD8, and CD3 expression at different time points. CD4 + CD8 -CD3 -lymphoid progenitor cells that could differentiate into mature T lymphocytes were observed after 15 days when a cocktail of cytokines, including Flt-3 ligand, stem cell factor, interleukin (IL)-12, and IL-2, was added. Results: These results thus show that CD4 + CD8 -CD3 -cells can be derived from CD34 + cells in vitro when cultured on TSCs. Conclusions: We showed that CD4 + CD8 -CD3 -cells can be derived from highly purified CD34 + cells on TSCs during T-cell lymphopoiesis in vitro.