Hematopoietic progenitor cells from children with Fanconi anemia of the C complementation group (FA-C) are excessively apoptotic and hypersensitive to various extracellular cues including Fas-ligand, tumor necrosis factor-α and double-stranded RNA. Interferon (IFN)-γ is known to augment apoptotic responses of these factors. The “priming” effect of IFN-γ is not fully explained. In view of the strong evidence that FA cells are intolerant of oxidative stress, we tested the notion that IFN-priming involves the induction of reactive oxygen species (ROS) in two FA-C B-lymphocyte cell lines and in peripheral blood neutrophils and mononuclear cells of FA patients. We also investigated whether the combination of IFN-γ and Fas created an intracellular environment that promoted apoptosis. Significantly lower doses of IFN-γ induced ROS accumulation in neutrophils and mononuclear cell of FA patients compared to cells of normal individuals. Enhanced ROS accumulation and decreased intracellular glutathione levels were observed in FA-C B-cell lines primed with IFN-γ and treated with agonistic anti-Fas antibody than in isogenic control cells corrected with FANCC. The above treatment also induced caspase-3 and -8 activation as well as apoptosis. That antioxidants reduced the priming effect of IFN-γ in Fas and IFN-γ-treated FA lymphoblast cells, demonstrates that ROS represent a critical effector mechanism for the exaggerated responses to IFN-γ characteristic of FA-C cells.