Abstract Notch signaling is implicated in stem cell self-renewal, differentiation, and other developmental processes, and the Drosophila hairy and enhancer of split (HES) 1 basic helix-loop-helix protein is a major downstream effector in the Notch pathway. We found that HES1 was expressed at high levels in the hematopoietic stem cell (HSC)–enriched CD34+/[CD38/Lin]− /low subpopulation but at low levels in more mature progenitor cell populations. When CD34+ cells were cultured for 1 week, the level of HES1 remained high in the CD34+ subset that had remained quiescent during ex vivo culture but was reduced in CD34+ cells that had divided. To investigate the effects of HES1 in human and mouse hematopoietic stem–progenitor cells (HSPCs), we constructed conditional lentiviral vectors (lentivectors) to introduce transgenes encoding either wild-type HES1 or a mutant lacking the DNA-binding domain (ΔBHES1). We found that lentivector-mediated HES1 expression in CD34+ cells inhibited cell cycling in vitro and cell expansion in vivo, associated with upregulation of the cell cycle inhibitor p21cip1/Waf1 (p21). The HES1 DNA–binding domain was required for these actions. HES1 did not induce programmed cell death or alter differentiation in HSPCs, and while short-term repopulating activity was reduced in HES1-transduced mouse and human cells, long-term reconstituting HSC function was preserved. Our data characterize the complex, cell context–dependent actions of HES1 as a major downstream Notch signaling regulator of HSPC function.