Two distinct bone marrow-derived blast colony-forming cells can generate colonies of lineage-restricted progenitor cells in agar cultures of murine bone marrow. Both cell types selectively had a Kit(+) ScaI(+) phenotype distinguishing them from most lineage-restricted progenitor cells. Multicentric blast colony-forming cells stimulated by stem cell factor plus interleukin-6 (IL-6) (BL-CFC-S) were separable from most dispersed blast colony-forming cells stimulated by Flt3 ligand and IL-6 (BL-CFC-F) using CD34 and Flt3R probes. Multicentric BL-CFC-S cofractionated with colony-forming units, spleen (CFU-S) supporting the possibility that the 2 cells may be identical. The colony populations generated by BL-CFC-S were similar in their phenotype and proliferative capacity to progenitor cells in whole bone marrow but the progeny of BL-CFC-F were skewed with an abnormally high proportion of Kit(-) Flt3R(+) cells whose clonogenic cells tended to generate only macrophage progeny. Both blast colony populations had a high percentage of GR1(+) and Mac1(+) cells but BL-CFC-F colonies also contained a significant population of B220(+) and IL-7R(+) cells relevant to the superior ability of BL-CFC-F colony cells to generate B lymphocytes and the known dependency of this process on Flt3 ligand and IL-7. The commitment events and phenotypic changes during the generation of differing progenitor cells in blast colonies can now be clonally analyzed in a convenient in vitro culture system.