Understanding the pathways that signal T cell tolerance versus activation is key to regulating immunity. Previous studies have linked CD28 and protein kinase C-θ (PKCθ) as a potential signaling pathway that influences T cell activation. Therefore, we have compared the responses of T cells deficient for CD28 and PKCθ in vivo and in vitro. Here, we demonstrate that the absence of PKCθ leads to the induction of T cell anergy, with a phenotype that is comparable to the absence of CD28. Further experiments examined whether PKCθ triggered other CD28-dependent responses. Our data show that CD4 T cell–B cell cooperation is dependent on CD28 but not PKCθ, whereas CD28 costimulatory signals that augment proliferation can be uncoupled from signals that regulate anergy. Therefore, PKCθ relays a defined subset of CD28 signals during T cell activation and is critical for the induction of activation versus tolerance in vivo.