Cytotoxic CD8+ T cells are key players of the adaptive immune defense against intracellular pathogens. Self-reactive CD8+ T cells harbour the potential to mount self-destructive responses, often resulting in severe autoimmune disease. However, the factors which cause self-destructive T cell responses remain poorly understood. The outcome of a CD8+ T cell response is determined by transcriptional regulators that influence T cell differentiation, effector function and memory formation. To unravel the mechanisms that drive self-reactive CD8+ T cell responses in the central nervous system (CNS), we established an in vivo RNA interferenc screening approach in a murine model of CD8+ T cell-driven neuroinflammation. This approach is based on retroviral delivery of short hairpin RNAs, to study, in a pooled fashion, the effect of distinct gene knock-downs on a cell's function. Using this approach, we could identify multiple transcription factors with a potential role in CD8+ T cell-mediated responses in the CNS.