Acquired aplastic anemia is characterized by loss or dysfunction of hematopoietic stem and progenitor cells. The proinflammatory cytokines Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) may be responsible for the immune-mediated pathology observed in some patients. The CD34+ population of bone marrow mononuclear cells contains primitive cells responsible for hemopoiesis. We investigated the response of CD34+ cells from aplastic anemia patients to a combination of IFN-gamma and TNF-alpha, and compared them to cells from normal volunteer donors. This was to determine whether aplastic CD34+ cells are more sensitive than normal cells to IFN-gamma/TNF-alpha-mediated effects, and whether cytokine-induced CD95 expression can explain the high levels of apoptosis observed in CD34+ cells from aplastic patients. CD34+38- cells were most affected by overnight incubation with these cytokines, their proportion and numbers being reduced in both normal donors and patients. There was no evidence for increased apoptosis, suggesting that this effect may be due to differentiation. IFN-gamma/TNF-alpha induced upregulation of CD95 on both normal and aplastic CD34+ cells, although the basal level of CD95 expression was increased in aplastic cells. However, CD95 induction did not make cells from normal donors or aplastic anemia patients susceptible to induction of apoptosis by agonistic anti-CD95 antibodies, soluble CD95 ligand, or membrane-bound CD95L. In vivo CD95L is required for CD95 induced apoptosis. No forms of this protein were detectable in lymphocytes from aplastic patients. We conclude that increased apoptosis in aplastic CD34+ cells is not due to increased sensitivity to IFN-gamma/TNF-alpha. We further show that normal and aplastic CD34+ cells are resistant to CD95 apoptosis, even in the presence of mCD95L.