The cell-cycle status of hematopoietic stem cells (HSCs) is tightly regulated, most likely to balance maintenance of stem-cell status through quiescence and expansion/differentiation of the hematopoietic system. Tumor-suppressor genes (TSGs), with their cell cycle-regulatory functions, play important roles in HSC regulation. The cyclin-D binding myb-like transcription factor 1 (Dmtf1) was recently recognized as a TSG involved in human cancers by repressing oncogenic Ras/Raf signaling. However, the role of Dmtf1 in the hematopoietic system is entirely unknown. In the present study, we demonstrate that Dmtf1 regulates HSC function under both steady-state and stress conditions. Dmtf1(-/-) mice showed increased blood cell counts in multiple parameters, and their progenitor cells had increased proliferation and accelerated cell-cycle progression. In addition, long-term HSCs from Dmtf1(-/-) mice had a higher self-renewal capacity that was clearly demonstrated in secondary recipients in serial transplantation studies. Dmtf1(-/-) BM cells showed hyper proliferation after 5-fluorouracil-induced myeloablation. Steady-state expression and Induction of CDKN1a (p21) and Arf were impaired in HSCs from Dmtf1(-/-) mice. The function of Dmtf1 was mediated by both Arf-dependent and Arf-independent pathways. Our results implicate Dmtf1 in the regulation of HSC function through novel cell cycle-regulatory mechanisms.