Gain-of-function experiments have demonstrated the potential of Notch signals to expand primitive hematopoietic progenitors, but whether Notch physiologically regulates hematopoietic stem cell (HSC) homeostasis in vivo is unclear. To answer this question, we evaluated the effect of global deficiencies of canonical Notch signaling in rigorous HSC assays. Hematopoietic progenitors expressing dominant-negative Mastermind-like1 (DNMAML), a potent inhibitor of Notch-mediated transcriptional activation, achieved stable long-term reconstitution of irradiated hosts and showed a normal frequency of progenitor fractions enriched for long-term HSCs. Similar results were observed with cells lacking CSL/RBPJ, a DNA-binding factor that is required for canonical Notch signaling. Notch-deprived progenitors provided normal long-term reconstitution after secondary competitive transplantation. Furthermore, Notch target genes were expressed at low levels in primitive hematopoietic progenitors. Taken together, these results rule out an essential physiological role for cell- autonomous canonical Notch signals in HSC maintenance