We examined the requirement for and cooperation between CD28 and inducible costimulator (ICOS) in effective T helper (T H) cell responses in vivo. We found that both CD28 and ICOS were critical in determining the outcome of an immune response; cytolytic T lymphocyte–associated antigen 4–immunoglobulin (CTLA-4–Ig), ICOS-Ig and/or a neutralizing ICOS monoclonal antibody attenuated T cell expansion, T H 2 cytokine production and eosinophilic inflammation. CD28-dependent signaling was essential during priming, whereas ICOS–B7RP-1 regulated T H effector responses, and the up-regulation of chemokine receptors that determine T cell migration. Our data suggests a scenario whereby both molecules regulate the outcome of the immune response but play separate key roles: CD28 primes T cells and ICOS regulates effector responses.