Human hematopoietic stem cells (HSCs) exposed to cytokines in vitro rapidly divide and lose their characteristic functional properties presumably due to the alteration of a genetic program that determines the properties of an HSC. We have attempted to reverse the silencing of this HSC genetic program by the sequential treatment of human cord blood CD34⁺ cells with the chromatin-modifying agents, 5-aza-2′-deoxycytidine (5azaD) and trichostatin A (TSA). We determined that all CD34⁺CD90⁺ cells treated with 5azaD/TSA and cytokines after 9 days of incubation divide, but to a lesser degree than cells exposed to only cytokines. When CD34⁺CD90⁺ cells that have undergone extensive number of cell divisions (5-10) in the presence of cytokines alone were transplanted into immunodeficient mice, donor cell chimerism was not detectable. By contrast, 5azaD/TSA-treated cells that have undergone similar numbers of cell divisions retained their marrow repopulating potential. The expression of several genes and their products previously implicated in HSC self-renewal were up-regulated in the cells treated with 5azaD/TSA as compared to cells exposed to cytokines alone. These data indicate that HSC treated with chromatin-modifying agents are capable of undergoing repeated cell divisions in vitro while retaining their marrow-repopulating potential.