TCR-microclusters (MC) are generated upon TCR stimulation prior to the immune synapse formation independently of lipid rafts. TCR-MCs contain receptors, kinases and adaptors, and function as the signaling unit for T cell activation. The TCR complex, but not the signaling molecules, is transported to the center to form cSMAC. The co-stimulation receptor CD28 joins the signaling region of cSMAC and recruits PKCθ and Carma1. CTLA-4 accumulates in the same region and competes with CD28 for negative regulation of T cell activation. T cell activation is therefore mediated by two spatially distinct signaling compartments: TCR signaling by the peripheral TCR-MC and co-stimulation signal by the central signaling cSMAC.