The present studies evaluated effects of recombinant human (rhu) and murine (rmu) flt3 ligand (flt3-L) on colony formation by subsets of myeloid stem and progenitor cells present in low-density (LD) and cell-sorted CD34 hu cord blood (CB) and bone marrow (BM) cells and unseparated mu BM cells. By itself, flt3-L had weak colony-stimulating activity. It stimulated small dispersed CFU-GM-type colonies, but not BFU-E, CFU-GEMM, or HPP-CFC colonies, from LD and CD34 huCB and BM. However, flt3-L had additive to greater-than-additive effects on colony number and size by CFU-GM stimulated with GM-CSF or IL-3, with or without Steel factor (SLF); by CFU-G stimulated by G-CSF with or without SLF; by CFU-M stimulated by CSF-1; and by BFU-E, CFU-GEMM, and HPP-CFC stimulated by Epo with or without IL-3 or SLF. Flt3-L enhanced the effects of SLF, alone and in combination with other CSFs. Similar effects were apparent on LD and sorted CD34 cells and also at the level of single sorted and isolated CD34 cells/well. Flt3-L enhanced expansion of immature subsets of huCD34(+)-column separated CB CFU-GM stimulated by the potent combination of SLF and PIXY321 (a GM-CSF/IL-3 fusion protein). While flt3-L did not enhance the replating capacity of CFU-GEMM plated in the presence of Epo and SLF, it enhanced numbers of these CFU-GEMM colonies with the capacity to be replated. Flt3-L effects were not species-specific; rhu and rmu forms were active on huCB/BM and muBM. These results demonstrate the potent direct-acting stimulating/costimulating activities of flt3-L in vitro on myeloid stem/progenitor cells.