Generation of CD8+ T cell immunity depends on costimulatory signals delivered by fully mature antigen-presenting dendritic cells (DCs), which are activated by pathogen-associated molecular patterns (PAMPs) through pattern-recognition receptors (PRR). In contrast, immature DCs contribute to T cell tolerance by delivery of coinhibitory signals. Liver sinusoidal endothelial cells (LSEC) are unique organ-resident antigen-presenting cells that mediate CD8+ T cell tolerance similar to immature DCs. We addressed the question whether LSEC undergo functional maturation after contact with PAMPs or viral infection. We demonstrate that LSEC express numerous PRR that attribute sentinel function. However, LSEC did not undergo maturation in response to ligands for PRR as they failed to up-regulate known co-stimulatory molecules and still induced tolerance in naïve CD8+ T cells. In contrast, infection with the murine cytomegalovirus led to functional maturation of LSEC by cell-autonomous mechanisms promoting differentiation of naïve CD8+ T cells into IFNγ-producing effector cells. Most surprising, classical costimulatory signals were not involved in this process. Genome-wide transcriptional analysis of LSEC revealed fundamental differences to gene expression in matured DCs. We identified unique gene patterns specifically regulated in immunogenic LSEC but not DCs showing alternative principles for induction of CD8 T cell differentiation. Our results demonstrate that CD8 T cell differentiation can occur in the absence of well-known costimulatory signals through liver-resident endothelial cells licensed by viral infection. These results shed new light on the mechanisms of organ-specific autoimmunity and may help to improve the efforts to overcome tolerance in relevant situations such as cancer.