Exposure of murine bone marrow (BM) cells to ionizing radiation (IR; 4 Gy) resulted in >95% inhibition of the frequency of various day types of cobblestone area-forming cells in association with the induction of apoptosis in hematopoietic stem cell alike cells (Lin⁻ ScaI⁺ c-kit⁺ cells; IR: 64.8 ± 0.4% versus control: 20.4 ± 0.5%; P < 0.001) and progenitors (Lin⁻ ScaI⁻ c-kit⁺ cells; IR: 46.2 ± 1.4% versus control: 7.8 ± 0.5%; P < 0.001). Incubation of murine BM cells with busulfan (BU; 30 μm) for 6 h also inhibited the cobblestone area-forming cell frequency but failed to cause a significant increase in apoptosis in these two types of hematopoietic cells. After 5 weeks of long-term BM cell culture, 33% and 72% of hematopoietic cells survived IR- and BU-induced damage, respectively, as compared with control cells, but they could not form colony forming units-granulocyte macrophages. Moreover, these surviving cells expressed an increased level of senescence-associated β-galactosidase, p16^Ink4a, and p19^Arf. These findings suggest that IR inhibits the function of hematopoietic stem cell alike cells and progenitors primarily by inducing apoptosis, whereas BU does so mainly by inducing premature senescence. In addition, induction of premature senescence in BM hematopoietic cells also contributes to IR-induced inhibition of their hematopoietic function. Interestingly, the induction of hematopoietic cell senescence by IR, but not by BU, was associated with an elevation in p53 and p21^Cip1/Waf1 expression. This suggests that IR induces hematopoietic cell senescence in a p53-p21^Cip1/Waf1-dependent manner, whereas the induction of senescence by BU bypasses the p53-p21^Cip1/Waf1 pathway.