CD28 costimulation regulates a wide range of cellular processes, from proliferation and survival to promoting the differentiation of specialized T-cell subsets. Since first being identified over 20 years ago, CD28 has remained a subject of intense study because of its profound consequences on T cell function and its potential for therapeutic manipulation. In this review we highlight the signaling cascades initiated by the major signaling motifs in CD28, focusing on PI-3 kinase-dependent and -independent pathways and how these are linked to specific cellular outcomes. Recent studies using gene targeted knockin mice have clarified the relative importance of these motifs on in vivo immune responses; however, much remains to be elucidated. Understanding the mechanism behind costimulation holds great potential for development of new clinically relevant reagents, a fact beginning to be realized with the advent of drugs that prevent CD28 ligation and signaling.