The immunological synapse formed during mature T cell activation consists of a central cluster of TCR and MHC molecules surrounded by a ring of LFA-1 and ICAM-1. We examined synapse formation in thymocytes undergoing activation in a lipid bilayer system by following the movement of fluorescent MHC and ICAM-1 molecules. Immature CD4+CD8+ thymocytes formed a decentralized synapse with multiple foci of MHC accumulation corresponding to areas of exclusion of ICAM-1. The MHC clusters and ICAM-1 holes were mobile and transient and correlated with active and sustained signaling, as shown by staining with antibodies against phosphotyrosine and activated Lck. Our findings show that signaling in immature thymocytes can result from a novel, multifocal pattern of receptor accumulation.