T cell activation begins with the formation of signaling complexes at the cell surface involving the T cell antigen receptor (TCR), the Src family kinase Lck and the adaptor protein, linker for activation of T cells (LAT). How early TCR signaling events are regulated to prevent inopportune signaling in resting T cells but efficient activation upon receptor ligation is poorly understood. We have established super-resolution fluorescence imaging approaches known as localization microscopy to determine how TCR engagement reorganizes signaling proteins on the molecular scale. Imaging single molecules in intact cells has provided new insights into the mechanisms of Lck clustering (Rossy et al. Nat Immunol 2012) and LAT recruitment (Williamson et al. Nat Immunol 2011) upon TCR activation. We are now extending this work to elucidate how the membrane environment affects protein interactions (Owen et al. Nat Commun 2012) to better understand the molecular principles of TCR signaling regulation.