Since tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and transforming growth factor (TGF)-beta have all been shown to be specific inhibitors of early human hematopoiesis, we wanted to investigate the interactions of these three cytokines on very primitive human adult bone marrow CD34++CD38- hematopoietic progenitor cells, using a pre-colony-forming cell (pre-CFC) assay, which detects the effects of these cytokines on the initial phases of the differentiation of these primitive progenitors, which are unresponsive to interleukin (IL) 3 alone. Surprisingly, TNF-alpha was a very potent stimulator of the proliferation of CD34++CD38- cells and was the most potent synergistic factor for the IL-3-induced proliferation of these cells of all cytokines tested (IL-1, IL-6, granulocyte colony-stimulating factor, kit ligand). TNF-alpha was the only cytokine that, as a single added factor, induced substantial proliferation in CD34++CD38- cells in the presence of IL-3, except for kit ligand, which induced very limited proliferation. TNF-alpha, moreover, induced a high degree of resistance to the inhibitory effects of TGF-beta in a dose-dependent way. The inhibitory effects of IFN-gamma, however, were not affected by the presence of TNF-alpha. We hypothesize that in situations of the hematopoietic stress, TNF-alpha may abrogate the inhibitory effect of ambient TGF-beta in the bone marrow microenvironment to allow primitive stem cells to proliferate and differentiate in response to an increased demand for mature blood cells.