An evaluation of the effects of a recombinant, soluble form of the c-kitligand alone and in combination with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) on the regulation of human megakaryocytopoiesis was performed using a serum-depleted clonal assay system and a long-term bone marrow culture system. The effects of thec-kitligand on the primitive megakaryocyte (MK) progenitor cell, the burst-forming unit-megakaryocyte (BFU-MK), and the more differentiated colony-forming unit-megakaryocyte (CFU-MK) were determined. Thec-kitligand alone had no megakaryocyte colony-stimulating activity (MK-CSA) but was capable of augmenting the MK-CSA of both GM-CSF and IL-3. The range of synergistic interactions ofc-kitligand varied with the class of MK progenitor cell assayed. In the case of the BFU-MK, thec-kitligand synergistically augmented the numbers of colonies formed in the presence of IL-3, but not GM-CSF, but increased the size of BFU-MK–derived colonies cloned in the presence of both of these cytokines. However, at the level of the CFU-MK,c-kitligand synergized with both GM-CSF and IL-3 by increasing both colony numbers and size. Although thec-kitligand alone exhibited limited potential in sustaining long-term megakaryocytopoiesis in vitro, it syngeristically augmented the ability of IL-3, but not GM-CSF, to promote long-term megakaryocytopoiesis. These data indicate that multiple cytokines are necessary to optimally stimulate the proliferation of both classes of MK progenitor cells and that thec-kit ligand plays a significant role in this process by amplifying the MK-CSA of both GM-CSF and IL-3.