Hematopoietic cells are often exposed to transient hypoxia and reoxygenation as they develop and migrate. Given that bone marrow (BM) failure occurred in patients with Fanconi anemia (FA), we reason that hypoxia-then-reoxygenation represents a physiologically relevant stress for FA hematopoietic progenitor/stem cells. Here we show that expansion of Fancc–/– BM cells enriched for progenitor and stem cells was significantly decreased after 2 continuous cycles of hyperoxic-hypoxic-hyperoxic treatments compared with wild-type (WT) BM cells. This inhibition was attributable to a marked decrease of lineage-depleted (Lin–) ScaI– c-kit+ cells and more primitive Lin– ScaI+ c-kit+ cells in Fancc–/– BM cells following reoxygenation. Evaluation of the cell-cycle profile of long-term BM culture (LTBMC) revealed that a vast majority (70.6%) of reoxygenated Fancc–/– LTBMC cells was residing in the G0 and G1 phases compared with 55.8% in WT LTBMC cells. Fancc–/– LTBMC cells stained intensely for SA-β-galactosidase activity, a biomarker for senescence; this was associated with increased expression of senescence-associated proteins p53 and p21WAF1/CIP1. Taken together, these results suggest that reoxygenation induces premature senescence in Fancc–/– BM hematopoietic cells by signaling through p53, up-regulating p21, and causing senescent cell-cycle arrest. Thus, reoxygenation-induced premature senescence may be a novel mechanism underlying hematopoietic cell depletion and BM failure in FA.