We analyzed the avidity and CD28-mediated co-stimulatory requirements for the activation of T cells in vivo and in vitro. The strength of the T cell/antigen-presenting cell interaction was varied by using altered peptide ligands for stimulation. Co-stimulatory requirements were studied using T cells from CD28-deficient mice. The results indicate that T cell activation is not an all-or-nothing event, but occurs in distinct steps. For each step, a certain avidity, co-stimulatory threshold or both, must be met. Depending upon the strength of the interaction between the T cell receptor and the major histocompatibility complex/peptide and the presence of CD28 co-stimulatory signals, T cells may undergo blast formation alone or proliferate or eventually both proliferate and differentiate to effector cells. Thus, T cell activation is governed by both avidity and co-stimulatory thresholds.