Exposure to ionizing radiation (IR) and certain chemotherapeutic agents not only causes acute bone marrow (BM) suppression but also leads to long-term residual hematopoietic injury. This latter effect has been attributed to damage to hematopoietic stem cell (HSC) self-renewal. Using a mouse model, we investigated whether IR induces senescence in HSCs, as induction of HSC senescence can lead to the defect in HSC self-renewal. It was found that exposure of C57BL/6 mice to a sublethal dose (6.5 Gy) of total body irradiation (TBI) resulted in a sustained quantitative and qualitative reduction of LKS⁺ HSCs. In addition, LKS⁺ HSCs from irradiated mice exhibited an increased expression of the 2 commonly used biomarkers of cellular senescence, p16^Ink4a and SA-β-gal. In contrast, no such changes were observed in irradiated LKS^- hematopoietic progenitor cells. These results provide the first direct evidence demonstrating that IR exposure can selectively induce HSC senescence. Of interest, the induction of HSC senescence was associated with a prolonged elevation of p21^Cip1/Waf1, p19^Arf, and p16^Ink4a mRNA expression, while the expression of p27^Kip1 and p18^Ink4c mRNA was not increased following TBI. This suggests that p21^Cip1/Waf1, p19^Arf, and p16^Ink4a may play an important role in IR-induced senescence in HSCs.