The hematopoietic stem cell (HSC) is defined as a cell that can either self-replicate or generate daughter cells that are destined to commit to mature cells of different specific lineages. Self-replication of the most primitive HSC produces daughter cells that possess a long (possibly unlimited) clonal lifespan, whereas differentiation of HSC produces daughter cells that demonstrate a progressive reduction of their clonal lifespan, a loss of multilineage potential, and lineage commitment. Previous studies indicated that the proliferation of HSC ex vivo favors differentiation at the expense of self-replication, eventually resulting in a complete loss of HSC. In contrast, transplantation studies have shown that a single HSC can repopulate the marrow of a lethally irradiated mouse, demonstrating that self-renewal of HSC occurs in vivo. Thrombopoietin (TPO) has been shown to function both as a proliferative and differentiative factor for megakaryocytes and as a survival and weakly proliferative factor for HSC. Our studies focused on the effects of exogenous TPO on HSC in mouse long-term bone marrow cultures (LTBMC). Previous results indicate that HSC decline in LTBMC in the absence of TPO. In contrast, the continuous presence of TPO resulted in the generation of both long- and short-term repopulating HSC as detected by an in vivo competitive repopulation assay. HSC were generated over a 4-month period at concentrations similar to normal bone marrow. Our results demonstrate that TPO can mediate the self-replication of HSC in LTBMC, and provide proof that HSC can self-replicate ex vivo.