The formation of the immunological synapse between T cells and antigen-presenting cells (APC) begins within minutes of contact and can take hours for full T-cell activation. Although early phases of the synapse have been extensively studied for a select number of proteins, later phases have not yet been examined in detail. We studied the signaling network in stable synapses by measuring the simultaneous localization of 25 signaling and structural molecules over 2 h at the level of individual synapses using multi-epitope ligand cartography (MELC). Signaling proteins including phospho(p)ZAP70, pSLP76, pCD3, and pLAT, along with proteins that influence synapse structure such as F-actin, tubulin, CD45, and ICAM-1, were localized in images of synapses and revealed the multidimensional construction of a mature synapse. The construction of the stable synapse included intense early TCR signaling, a phase of recruitment of structural proteins, and a sustained increase in signaling molecules and colocalization of TCR and pLAT signaling clusters in the center of the synapse. Consolidation of TCR and associated proteins resulted in formation of a small number of discrete synaptic microclusters. Development of synapses and cSMAC composition was greatly affected by the absence of Vav1, with an associated loss in PLCâ„1 recruitment, pSLP76, and increased CXCR4. Adaptive immune responses are initiated by the meeting of a T cell and an antigen-presenting cell (APC) 1 bearing peptide-MHC (pMHC) complexes that are a specific fit for the T-cell receptor (TCR) on the T-cell surface. Within seconds, TCR signaling starts with a sequence of phosphorylation and de-phosphorylation events of membrane-proximal and -distal TCR-signaling molecules and their spatial reorganization into protein multiclusters (1). Together with the rearrangement of structural molecules at the cell-cell interface, these signals lead to the formation of a supramolecular structure termed the immunological synapse (1-3). The synapse can differ substantially in size and composition, but comprises several common structural motifs (4 -6). In the classical synapse, these structural motifs are organized in domains that form a target