Increased expression of interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in bone marrow failure disorders suggests a possible pathophysiologic role of these cytokines in disease. In this study, we tested the action of TNF-α and IFN-γ on phenotypically and functionally defined stages of hematopoietic development using highly purified progenitor cell populations assayed in standardized culture systems. We hypothesized that the inhibitory effects of IFN-γ and TNF-α might be related to the induction of programmed cell death. In methylcellulose colony assays, IFN-γ and TNF-α inhibited the growth of early hematopoietic cells, including committed CD34+ CD38+ progenitor cells and phenotypically less mature CD34+CD38− cells, with 50% decreased colony formation occurring in the range of 750-1,000 U/ml of IFN-γ and 10–15 ng/ml of TNF-α. More potent suppressive effects were observed in cultures supplemented with the combination of both cytokines than in cultures treated with IFN-γ or TNF-α alone. When used at these concentrations, IFN-γ and TNF-α inhibited growth of CD34+-enriched long-term culture-initiating cells by 88% and 68%, respectively. IFN-γ and TNF-α triggered apoptosis of total bone marrow and CD34+ cells, recognized by the presence of a characteristic pattern of DNA degradation after low molecular weight DNA extraction, and by detection of apoptotic cells by the in situ terminal deoxynucleotidyl transferase assay. We speculate that chronic exposure of hematopoietic tissue to TNF-α and IFN-γ in vivo may result in broad depletion of the stem and progenitor cell pools. Death of these cells due to apotosis rather than transient inhibition of proliferation may be responsible for long-lasting hematologic consequences. © 1995 Wiley-Liss Inc.