Retroviral infections generate a strong activation and expansion of regulatory T cells (Tregs). In the Friend Retrovirus (FV) mouse model this induction of Tregs is dependent on the cytokines IL-2 and membrane-bound TNFa, which are produced by virus-specific effector T cells. Since it was described that retroviral infections induce several distinct populations of cytotoxic cells, e.g. CD8⁺ CTL, cytotoxic CD4⁺ T cells, and NK cells, we analyzed the suppressive potential of Tregs on these different cell subsets. To this end we selectively depleted Tregs in FV-infected DEREG mice. We found that virus-induced Tregs suppressed CD8⁺ CTL responses during acute and chronic FV infection. Temporary depletion of Tregs resulted in reactivation of exhausted CD8⁺ T cells and a significant reduction of chronic viral set points. Cytotoxic CD4⁺ T cell responses were not only under the control of Tregs but also restricted by CD8⁺ T cells. Only a simultaneous depletion of Tregs and CD8⁺ T cells during acute infection resulted in a massive expansion of FV-specific CD4⁺ T cells that could kill FVinfected target cells in vivo. Anti-retroviral NK cell responses were also suppressed by activated Tregs. Treg depletion mediated NK cell activation and maturation and enhanced their cytotoxic potential during acute FV infection. Interestingly, Tregs suppressed NK cell responses by massive consumption of IL-2, which NK cells need for full activation. Treg suppression of NK cells could be overcome by a specific NK cell directed IL-2 therapy. The data demonstrate how important Tregs are in restricting cytotoxic responses against retrovirus infected cells and suggest novel therapeutic approaches based on Treg manipulation.