Neoplastic CD34+ cells from chronic myeloid leukemia (CML) patients proliferate in vitro in the absence of serum or defined growth factors due to an autocrine mechanism involving IL-3 and G-CSF (Jiang et al. Proc Natl Acad Sci USA 1999; 96: 12804). Detailed examination of the various cell types produced in such cultures has now demonstrated the rapid, factor-independent, generation of clonogenic progenitors for all lineages (granulocyte-macrophage, megakaryocyte and erythroid) with the additional appearance within 10 days of large numbers of mature granulocytes, macrophages, and megakaryocytes, as well as occasional erythroid cells. Inclusion of flt3-ligand, Steel factor, IL-3, IL-6, and G-CSF +/- erythropoietin (EPO) in the cultures enhanced only slightly the output of mature cells (except for the erythroid population which was much larger when EPO was added). Analogous subpopulations of normal CD34+ cells produced similar numbers and types of cells but, as expected, only when growth factors were added. Thus primitive CD34+ CML cells proliferating autonomously in vitro recapitulate the full spectrum of differentiation responses of normal CD34+ cells stimulated by IL-3 and G-CSF. These findings point to a role of autocrine IL-3 and G-CSF in the similar multi-lineage expansion of differentiating CD34+ CML cells that occurs in vivo.