The in vivo differentiation/survival of CD4⁺CD25⁺ T suppressor cells is dependent on IL‐2 and CD28‐mediated costimulatory signals. To determine the cytokine and costimulatory requirements for CD25⁺ T cells in vitro, we established a two‐stage culture system where CD25⁺ T cells were activated in a primary culture. In the subsequent culture, activated CD4⁺CD25⁺ cells were then mixed with responders in order to assess their suppressor function. Pre‐culture of CD25⁺ T cells with anti‐CD3 alone resulted in poor survival and minimal induction of suppressor activity. Pre‐culture in the presence of anti‐CD3 and IL‐2 or IL‐4, but not IL‐6, IL‐7, IL‐9, IL‐10 or IL‐15, resulted in proliferation of the CD25⁺ cells and induction of potent suppressor function. Inhibition of the interaction of CD28 or cytotoxic T lymphocyte‐associated antigen‐4 (CTLA‐4) with CD80/CD86 in the pre‐culture of CD4⁺CD25⁺ cells did not prevent the induction of suppressor function. Furthermore, the inhibition of costimulatory signals did not inhibit the ability of fresh CD25⁺ T cells to inhibit CD8⁺ responders under conditions where activation of the responders was independent of CD80/CD86. These studies support the view that activation of CD25⁺ T cells requires IL‐2/IL‐4 for their survival/differentiation into effector cells, but is independent of CD28/CTLA‐4‐mediated costimulation.