Inducible costimulator (ICOS) expression is critical for T cellmediated immunity. We showed previously that T cell receptor and CD28 coengagement up-regulate ICOS expression in activated T cells via the induction of NFATc2. Here, we examined the regulation of ICOS expression by Th-specific transcription factors T-bet and GATA-3. Overexpression of T-bet or GATA-3 alone could enhance, and NFATc2 could further synergize with either of them to increase, icos transcription. Although T-bet acted on the icos promoter, GATA-3 operated via an icos 3-untranslated region element. Interestingly, NFATc2 was found to bind promiscuously the icos promoter in developing Th0, Th1, and Th2 cells but became selectively associated with T-bet at the promoter and with GATA-3 at the 3-untranslated region in fully differentiated Th1 and Th2 cells, respectively. Collectively, our results reveal a temporally evolving circuit in which the nonselectively expressed NFATc2 cooperates with Th-restricted T-bet or GATA-3 to direct transcription of a costimulatory gene via distinct regulatory elements in different Th cells undergoing differentiation.