Cytomegalovirus (CMV) is a latent virus which causes chronic activation of the immune system. Here, we demonstrate that cytotoxic and pro-inflammatory CD4+CD28null T cells are only present in CMV seropositive donors and that CMV-specific Immunoglobulin (Ig) G titers correlate with the percentage of these cells. In vitro stimulation of peripheral blood mononuclear cells with CMVpp65 peptide resulted in the expansion of pre-existing CD4+CD28null T cells. In vivo, we observed de novo formation, as well as expansion of CD4+CD28null T cells in two different chronic inflammation models, namely the murine CMV (MCMV) model and the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis (MS). In EAE, the percentage of peripheral CD4+CD28null T cells correlated with disease severity. Pre-exposure to MCMV further aggravated EAE symptoms, which was paralleled by peripheral expansion of CD4+CD28null T cells, increased splenocyte MOG reactivity and higher levels of spinal cord demyelination. Cytotoxic CD4+ T cells were identified in demyelinated spinal cord regions, suggesting that peripherally expanded CD4+CD28null T cells migrate towards the central nervous system to inflict damage. Taken together, we demonstrate that CMV drives the expansion of CD4+CD28null T cells, thereby boosting the activation of disease-specific CD4+ T cells and aggravating autoimmune mediated inflammation and demyelination.