Hematopoietic stem cells (HSCs) have a very low rate of cell division in the steady state; however, under conditions of hematopoietic stress, these cells can begin to proliferate at high rates, differentiate into mature hematopoietic cells, and rapidly reconstitute ablated bone marrow (BM). Previously, we isolated a novel evolutionarily conserved DNA replication factor, PSF1 (partner of SLD5-1), from an HSC-specific cDNA library. In the steady state, PSF1 is expressed predominantly in CD34(+)KSL (c-kit(+)/Sca-1(+)/Lineage(-)) cells and progenitors, whereas high levels of PSF1 expression are induced in KSL cells after BM ablation. In 1-year-old PSF1(+/-) mice, the pool size of stem cells and progenitors is decreased. Whereas young PSF1(+/-) mutant mice develop normally, are fertile, and have no obvious differences in hematopoiesis in the steady state compared with wild-type mice, intravenous injection of 5-fluorouracil (5-FU) is lethal in PSF1(+/-) mice, resulting from a delay in induction of HSC proliferation during ablated BM reconstitution. Overexpression studies revealed that PSF1 regulates molecular stability of other GINS components, including SLD5, PSF2, and PSF3. Our data indicate that PSF1 is required for acute proliferation of HSCs in the BM of mice.