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Autoimmune hemolytic anemia (AIHA) is the result of increased destruction of red blood cells (RBCs) due to the production of autoantibodies, and it can be life-threatening. To study the mechanisms that trigger AIHA, we used the Marshall-Clarke and Playfair model of murine AIHA, in which mice repeatedly immunized with rat RBCs develop erythrocyte autoantibodies as well as rat-specific alloantibodies. We analyzed the role of CD25⁺ T-regulatory subsets in controlling AIHA in C57/Bl6 mice using antibody depletion studies. Treatment with anti-CD25 antibody but not isotype control prior to immunization with rat RBCs increased the incidence of AIHA from 30% to 90%. Adoptive transfer of purified splenic population of CD4⁺CD25⁺ but not CD4⁺CD25^- cells from immunized mice into naive recipients prevented the induction of autoantibody production. Altogether, our data establish a critical role for CD4⁺CD25 …