Ag recognition by T lymphocytes induces immune synapse formation and recruitment of signaling molecules into a lipid raft. Cbp/PAG is a Csk-associated membrane adapter protein exclusively localized in a lipid raft. We identified NHERF/EBP50 as a Cbp-binding molecule, which connects the membrane raft and cytoskeleton by binding to both Cbp through its PDZ domain and ezrin-radixin-moesin through the C terminus. Overexpression of Cbp reduced the mobility of the raft on the cell surface of unstimulated T cells and prevented synapse formation and subsequent T cell activation, whereas a mutant incapable of EBP50 binding restored both synapse formation and activation. These results suggest that anchoring of lipid raft to the cytoskeleton through Cbp-EBP50-ezrin-radixin-moesin assembly regulates membrane dynamism for synapse formation and T cell activation.