The structure of a T cell receptor (TCR) and its affinity for cognate antigen are fixed, but T cells regulate binding sensitivity through changes in lateral membrane organization. TCR microclusters formed upon antigen engagement participate in downstream signaling. Microclusters are also found 3-4 days after activation, leading to enhanced antigen binding upon rechallenge. However, others have found an almost complete loss of antigen binding four days after T cell activation, when TCR clusters are present. To resolve these contradictory results, we compared binding of soluble MHC-Ig dimers by transgenic T cells stimulated with a high (100 μM) or low (100 fM) dose of cognate antigen. Cells activated by a high dose of peptide bound sixfold lower amounts of CD8-dependent ligand K(b)-SIY than cells activated by a low dose of MHC/peptide. In contrast, both cell populations bound a CD8-independent ligand L(d)-QL9 equally well. Consistent with the differences between binding of CD8-dependent and CD8-independent peptide/MHC, Förster resonance energy transfer (FRET) measurements of molecular proximity reported little nanoscale association of TCR with CD8 (16 FRET units) compared to their association on cells stimulated by low antigen dose (62 FRET units). Loss of binding induced by changes in lateral organization of TCR and CD8 may serve as a regulatory mechanism to avoid excessive inflammation and immunopathology in response to aggressive infection