The human Lin⁻CD34⁻ cell population contains a newly defined class of hematopoietic stem cells that reconstitute hematopoiesis in xenogeneic transplantation systems. We therefore developed a culture condition in which these cells were maintained and then acquired CD34 expression and the ability to produce colony-forming cells (CFC) and SCID-repopulating cells (SRCs). A murine bone marrow stromal cell line, HESS-5, supports the survival and proliferation of Lin⁻CD34⁻ cells in the presence of fetal calf serum and human cytokines thrombopoietin, Flk-2/Flt-3 ligand, stem cell factor, granulocyte colony-stimulating factor, interleukin-3, and interleukin-6. Although Lin⁻CD34⁻ cells do not initially form any hematopoietic colonies in methylcellulose, they do acquire the colony-forming ability during 7 days of culture, which coincides with their conversion to a CD34⁺ phenotype. From 2.2% to 12.1% of the cells became positive for CD34 after culture. The long-term multilineage repopulating ability of these cultured cells was also confirmed by transplantation into irradiated NOD/SCID mice. These results represent the first in vitro demonstration of the precursor of CD34⁺ cells in the human CD34⁻ cell population. Furthermore, the in vitro system we reported here is expected to open the way to the precise characterization and ex vivo manipulation of Lin⁻CD34⁻ hematopoietic stem cells.