Mouse long-term hematopoietic reconstituting cells exist in the c-Kit⁺Sca-1⁺Lin⁻ (KSL) cell population; among them, CD34^low/− cells represent the most highly purified population of hematopoietic stem cells in the adult bone marrow. Here, we demonstrate that retrovirus-mediated transduction of CD34^low/−c-Kit⁺Sca-1⁺Lin⁻(34⁻KSL) cells with the HES-1 gene, which encodes a basic helix-loop-helix transcription factor functioning downstream of the Notch receptor, and is a key molecule for the growth phase of neural stem cells in the embryo, preserves the long-term reconstituting activity of these cells in vitro. We also show that cells derived from the HES-1–transduced 34⁻KSL population produce progenies characterized by negative Hoechst dye staining, which defines the side population, and by CD34^low/− profile in the bone marrow KSL population in each recipient mouse at ratios 3.5- and 7.8-fold those produced by nontransduced 34⁻KSL-derived competitor cells. We conclude that HES-1 preserves the long-term reconstituting hematopoietic activity of 34⁻KSL stem cells ex vivo. Up-regulation of HES-1 protein in the 34⁻KSL population before unnecessary cell division, that is, without retrovirus transduction, may represent a potent approach to absolute expansion of hematopoietic stem cells.