The objective of this study is to establish the aging model of murine hematopoietic stem cell (HSC) ex vitro and investigate its relative biological mechanism, aimed to build the foundation for searching the methods to delaying HSC aging. Sca-1(+)HSC were isolated and purified from murine bone marrow mono-nucleated cell by magnetic-activated cell sorting. The purity of separated cells was analyzed by flow cytometry (FCM) and the expression of Sca-1 was detected by immunofluorescence. Sca-1(+)HSC induced aging by tert-butylhydroperoxide (t-BHP, final concentration of 100 μmol/L) for 6 h to establish the murine HSC aging model in vitro. Biological characteristics of aging HSC were evaluated by mixed hematopoietic progenitor cell culture in vitro, cell cycle assay and senescence-associated β-galactosidase (SA-β-gal) cytochemical staining. Telomere length and telomerase activity were detected by southern blotting and telomere repeat amplification protocol-polymerase chain reaction (TRAP-PCR) augmentation. The expressions of p16(INK4a), P19(Arf), P53, P21(Cip1/Waf1) mRNA were detected by reverse transcription (RT)-PCR. The purity of separated Sca-1(+) HSC was 87.2% and the survival of Sca-1(+) HSC was 96~99%. After 6 h cocultured with 100 μmol/L t-BHP, the ability of aging Sca-1(+) HSC to form mixed hematopoietic progenitor colony, self-renewal and multi-differentiation were decreased significantly. The number of aging Sca-1(+) HSC entered G1 phase of the cell cycle and the percentage of positive cells expressed SA-β-gal increased significantly. The telomere length shortened and the telomerase activity decreased. The expression of p16(INK4a), p19(Arf), p53, P21(Cip1/Waf1) mRNA increased. The t-BHP can induce Sca-1(+) HSC senescence in vitro. The signal transduction pathways of p16(INK4a)-retinoblastoma and P19(Arf)-Mdm2-P53-P21(Cip1/Waf1) may play key roles in the Sca-1(+) HSC senescence induced by t-BHP.