CD4⁺CD25⁺ T cells regulate a variety of autoimmune and alloimmune responses including the development of autoimmune diabetes in non‐obese diabetic (NOD) mice. We have examined the role of CD28/CTLA4/B7 interactions in the expansion and survival of CD4⁺CD25⁺ regulatory T cells (T_reg) in this setting. CD28/B7 interactions are essential in the development of T_reg in the thymus and for their survival in the periphery. The CD28‐mediated homeostasis of these cells is independent of IL2, OX40, CD40L, and survival factor Bcl‐XL. In addition, analysis of T_reg from CTLA4‐deficient mice suggests that CTLA4 expression is not required for their development or function. However, non‐activating anti‐CTLA4 antibodies blocked the suppressor activity of regulatory cells in vitro. Thus, clinical application of co‐stimulatory blockade using agents such as CTLA4Ig in the treatment of autoimmune disease may result in complicated outcomes.