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.