Experimental cerebral malaria (ECM) is a gamma interferon (IFN- gamma)-dependent syndrome. However, whether IFN-gamma promotes ECM through direct and synergistic targeting of multiple cell populations or by acting primarily on a specific responsive cell type is currently unknown. Here, using a panel of cell- and compartment-specific IFN-gamma receptor 2 (IFN-gammaR2)-deficient mice, we show that IFN-gamma causes ECM by signaling within both the hematopoietic and nonhematopoietic compartments. Mechanistically, hematopoietic and nonhematopoietic compartment-specific IFN-gammaR signaling exerts additive effects in orchestrating intracerebral inflammation, leading to the development of ECM. Surprisingly, mice with specific deletion of IFN-gammaR2 expression on myeloid cells, T cells, or neurons were completely susceptible to terminal ECM. Utilizing a reductionist in vitro system, we show that synergistic IFN-gamma and tumor necrosis factor (TNF) stimulation promotes strong activation of brain blood vessel endothelial cells. Combined, our data show that within the hematopoietic compartment, IFN- gamma causes ECM by acting redundantly or by targeting non-T cell or non- myeloid cell populations. Within the nonhematopoietic compartment, brain endothelial cells, but not neurons, may be the major target of IFN-gamma leading to ECM development. Collectively, our data provide information on how IFN-gamma mediates the development of cerebral pathology during malaria infection.