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Introduction: After the onset of stroke, infiltrated CD8+ T cells aggravate brain injury through FasL or cytotoxic granules. We previously reported that FasL mutation could attenuate brain inflammation in experimental stroke, however, the exact mechanisms were undefined. Thus, this study tested the hypothesis whether FasL could modulate the cytotoxicity of CD8+ T cells or not.Methods: Middle cerebral artery occlusion was induced in both wide-type (WT) and FasL-mutant (gld) mice. Magnetic activated cell sorting was used to isolate CD8+ T cells from the spleen. In vitro, CD8+ T cells were cocultured with neurons or microglia for 24 hours. In some experiments, FasL neutralizing antibody were used to block FasL on CD8+ T cells. Flow cytometry and q-PCR were performed to measure the cytotoxicity of CD8+ T cells and polarization of microglia. Cytometric Bead Array was used to evaluate the inflammatory cytokines in the coculture supernatant. The cell viability of oxygen glucose deprivation (OGD) neurons was measured by lactate dehydrogenase assay and propidium iodide and calcein AM staining.Results: CD8+ T cells were activated and expressed more cytotoxic cytokines when cocultured with microglia. Nevertheless, this induction was attenuated when CD8+ T cells were pretreated with FasL neutralizing antibody. Meanwhile, FasL mutation or functional block abrogated CD8+ T cell-mediated killing of neurons and M1 microglia polarization. In addition, the CD8+ T cell-microglia coculture supernatant contained less pro-inflammatory cytokines and resulted in enhanced cell viability of post-OGD neuron when FasL on CD8+ T cells was mutant or blocked.Conclusions: FasL enhances cytotoxicity of CD8+ T cells after ischemic stroke.