Two Notch ligand families, Delta and Serrate/Jagged, have been identified in vertebrates. Members of the Jagged family have been shown to affect in vitro hematopoiesis. To determine whether members of the Delta family might play a similar role in hematopoiesis, we examined the expression of mouse Delta-like-1 (mDll1). mDll1 protein was detected in whole marrow and in a marrow stromal cell line MS-5. At the RNA level, both mDll1 and Notch1 were seen in marrow precursor, differentiated hematopoietic, marrow stromal, and MS-5 cells. We isolated a cDNA encoding the human homologue of mDll1, designated human Delta-like-1 (hDll1). A soluble form of hDll1, hDll1^NDSL, containing the DSL domain and the N-terminal sequences, was expressed and purified from bacteria as a glutathione S-transferase (GST) fusion protein. We observed that hDll1^NDSL delayed the acquisition of differentiation markers by murine hematopoietic progenitor cells (Lin⁻) cultured in vitro with cytokines. In addition, it promoted greater expansion (more than 3 times) of the primitive hematopoietic precursor cell population, measured in high-proliferative potential colony assay and day 12 colony-forming unit spleen (CFU-S) assay, than GST controls. We also observed that the percentage of apoptotic cells decreased and that the number of cells in the S-phase of the cell cycle increased in the cultures of Lin⁻cells with hDll1^NDSL. The effects of hDll1^NDSL were blocked by antibody against the mouse counterpart of hDll1^NDSL, mDll1^NDSL. These observations demonstrate that hDll1 plays a role in mediating cell fate decisions during hematopoiesis.