Closely related Hoechst 33342 (Ho) dull primitive hematopoietic stem cells (PHSC) subsetted on the basis of their increasing Rhodamine123 (Rh) uptake differ in transplant potential, cell cycling status, turnover time, and cytokine requirements for optimal growth in vitro. The finding that PHSC are not dormant, but cycle continuously throughout life, suggests that the proliferative capacity and homeostasis of the hematopoietic system must be regulated by either the selective control of cell death, or by the rate of proliferation or differentiation within the stem cell pool. We have applied semi-quantitative RT-PCR to compare the expression of c-myb, a transcription factor implicated in PHSC proliferation; and bcl-2, an apoptotic antagonist, in PHSC subsets in different states of activation or at various levels of maturity. The expression patterns of other genes implicated in regulation of stem cell maintenance, proliferation or differentiation was also determined. These include c-kit, c-jun, plk, mdr-1, mdr-2, bcl-xl, bcl-xs, bax, bad, and bak. Bcl-2 gene expression was detected in Hodull/Rhmedium PHSC, but not in the more primitive Hodull/Rhdull stem cells with the highest hematopoietic regenerative capacity. However, c-myb gene expression was higher in Hodull/Rhdull PHSC compared to Hodull/Rhmedium PHSC. The inverse correlation between bcl-2 gene expression, c-myb gene expression and stem cell potential suggests that bcl-2 expression is not c-myb dependent in early hematopoiesis. In vitro functional analysis of c-myb and bcl-2 knockout mice showed almost a complete absence of primitive stem cells in c-myb −/− fetal liver, and a 2 fold increase in the incidence of more committed progenitors in bcl-2 −/− bone marrow. Further gene expression analysis showed that Hodull/Rhdull PHSC expressed markedly elevated levels of c-kit, c-jun, mdr-I and mdr-2. This data provides an insight into positional effects of c-myb and bcl-2 within the stem cell hierarchy, suggesting that while c-myb is likely to be required to promote proliferation of the most primitive stem cells, bcl-2 may be responsible for homeostatic regulation of hematopoiesis through inhibition of proliferation and/or regulation of apoptosis of more mature primitive hematopoietic progenitor cells.