T cell activation begins with the interaction between an antigen-specific T cell and an antigen-presenting cell (APC). This interaction results in the formation of the immunological synapse, which had been considered to be responsible for antigen recognition and T cell activation. Recent advances in imaging analysis have provided new insights into T cell activation. The T cell receptor (TCR) microclusters, TCRs, kinases, and adaptors are generated upon antigen recognition at the interfaces between the T cells and the APCs and serve as a fundamental signaling unit for T cell activation. CD28-mediated costimulation is also found to be regulated by the formation of microclusters. Therefore, the dynamic regulations of TCR and CD28 microcluster formation, migration, and interaction are the key events for the initiation of T cell-mediated immune responses. Comprehensive analyses of the composition and characteristics of the TCR microcluster have identified its dynamic features. This review will outline new discoveries of the microclusters and its related concept in T cell activation.