We investigated the relationship between ICOS, CD28, CTLA-4, and IL-2 to gain a better understanding of this family of costimulatory receptors in the immune response. Using magnetic beads coated with anti-CD3 and varying amounts of anti-ICOS and anti-CTLA-4 Abs, we show that CTLA-4 ligation blocks ICOS costimulation. In addition to inhibiting cellular proliferation, CTLA-4 engagement prevented ICOS-costimulated T cells from producing IL-4, IL-10, and IL-13. Both an indirect and direct mechanism of CTLA-4's actions were examined. First, CTLA-4 engagement on resting cells was found to indirectly block ICOS costimulation by interferring with the signals needed to induce ICOS cell surface expression. Second, on preactivated cells that had high levels of ICOS expression, CTLA-4 ligation blocked the ICOS-mediated induction of IL-4, IL-10, and IL-13, suggesting an interference with downstream signaling pathways. The addition of IL-2 not only overcame both mechanisms, but also greatly augmented the level of cellular activation suggesting synergy between ICOS and IL-2 signaling. This cooperation between ICOS and IL-2 signaling was explored further by showing that the minimum level of IL-2 produced by ICOS costimulation was required for T cell proliferation. Finally, exogenous IL-2 was required for sustained growth of ICOS-costimulated T cells. These results indicate that stringent control of ICOS costimulation is maintained initially by CTLA-4 engagement and later by a requirement for exogenous IL-2.