Activated macrophage-conditioned medium (M-CM) induces megakaryocytic differentiation of HIMeg-1 cells. The megakaryocytic differentiation activity (MDA) is proteinaceous since it is susceptible to treatments by proteinases, heat, and reducing agents. MDA is not thrombopoietin (TPO) since (1) TPO alone or in conjunction with several other recombinant cytokines fails to induce any degree of HIMeg-1 cell differentiation; and (2) a neutralizing antibody against TPO or an antibody against the extracellular domain of c-mplis unable to abolish M-CM-induced CD41 expression on HIMeg-1 cells. Reverse transcriptase-mediated polymerase chain reaction shows that HIMeg-1 cells express c-mplbut not TPO. Additional neutralizing antibody studies suggest that MDA is not one of the cytokines known to induce some degree of megakaryopoiesis in vitro or in vivo including interleukin 3 (IL-3), IL-6, IL-11, granulocyte–macrophage colony-stimulating factor, erythropoietin, or stem cell factor. On the other hand, MDA appears to be a combination of interferon γ (IFN-γ) and tumour necrosis factor α (TNF-α), since neutralizing antibodies against these two cytokines completely abolish MDA-induced CD41 expression. In addition, either recombinant human IFN-γ or TNF-α alone is capable of inducing CD41 and CD42 expression on HIMeg-1 cells. In combination, IFN-γ and TNF-α induce a maximal level of CD41 and CD42 expression which is also accompanied by an increase in cell size and DNA ploidy level. Thus, our studies indicate that IFN-γ/TNF-α is capable of inducing megakaryocytic differentiation of the HIMeg-1 cell line and that HIMeg-1 is a good system for studying the molecular mechanism mediating megakaryocytic differentiation.