Spleen colony formation after transplantation of bone marrow cells into irradiated mice has been used as an assay for hematopoietic stem cells (CFU-S), but has serious limitations intrinsic to an in vivo assay. In this report we describe experiments using an in vitro clonogenic assay that is especially suitable for studies of stem cell regulation as defined growth factors and normal untreated bone marrow can be used. We have demonstrated that the colony-forming cells have proliferative properties in common with CFU-S and respond to specific proliferation regulators previously detected using the spleen colony assay.