The supernatant fluid of stimulated spleen cells (PHA-SCM) supported in vitro colony growth of murine marrow. In the absence of exogenous erythropoietin, it stimulated the growth of (1) myeloid colonies and (2) distinct mixed colonies containing erythroid cells, granulocytes, macrophages, and infrequently megakaryocytes in a setting structurally resembling biopsied marrow. The cells that form mixed colonies reside in a density range of 1.058–1.068 g/ml in a discontinuous albumin gradient. Active supernatant was produced by T cells in combination with a macrophage factor. DNA synthesis correlated with activity. PHA- SCM differed from erythropoietin (EPO) when chromatographed on lectin columns and did not contain EPO activity as demonstrated by the fetal mouse liver cell (FMLC) assay. The activity for mixed colony growth could be eluted from an anion exchange column with 0.07 M NaCl and eluted in a gel filtration column at a distance corresponding to a molecular weight of 39,000. Mixed colony-forming cells responsive to PHA-SCM were found to be Ia-H-2+. BFU-Es, CFU-Cs, and progenitors for myeloid colonies responsive to PHA-SCM were also H-2+ but showed significant sensitivity to anti-Ia antisera reflecting variable antigenic density. The mixed colony-forming cell appeared less differentiated than myeloid or erythroid progenitor cells examined, and its antigenic determinants are consistent with those observed for the pluripotent stem cell assayed in vivo (CFU-S).