Cytotoxic CD4+ CD28− T cells form a rare subset in human peripheral blood. The presence of CD4+ CD28− cells has been associated with chronic viral infections, but how these particular cells are generated is unknown. In this study, we show that in primary CMV infections, CD4+ CD28− T cells emerge just after cessation of the viral load, indicating that infection with CMV triggers the formation of CD4+ CD28− T cells. In line with this, we found these cells only in CMV-infected persons. CD4+ CD28− cells had an Ag-primed phenotype and expressed the cytolytic molecules granzyme B and perforin. Importantly, CD4+ CD28− cells were to a large extent CMV-specific because proliferation was only induced by CMV-Ag, but not by recall Ags such as purified protein derivative or tetanus toxoid. CD4+ CD28− cells only produced IFN-γ after stimulation with CMV-Ag, whereas CD4+ CD28+ cells also produced IFN-γ in response to varicella-zoster virus and purified protein derivative. Thus, CD4+ CD28− T cells emerge as a consequence of CMV infection.