Studies of hematopoietic stem cell (HSC)-derived dendritic cells (DCs) are often limited by the rarity of HSC. To facilitate the study of DCs, we have generated a novel cell line (CR1) by retroviral Notch(IC) gene transfer into Sca1(+)ckit(+)lin- HSC. CR1 cells proliferated in vitro in the presence of recombinant interleukin-3. They maintained an immature progenitor cell phenotype and an intact karyotype. In the presence of granulocyte-macrophage colony-stimulating factor or Flt3L, CR1 cells differentiated into myeloid and plasmacytoid DCs, respectively. Functionally, CR1 cells were comparable to primary bone-marrow-derived DCs with respect to Toll-like-receptor-mediated maturation, cytokine release and capacity to induce effective antitumor immunity. CR1 cells thus provide an elegant new cellular tool to study DC development, function and preclinical DC-based immunotherapies.