The nanoscale spatial arrangement of T cell receptor (TCR) ligands critically influences their activation potential in CD8+ T cells, yet a comprehensive understanding of the molecular landscape induced by engagement with native peptide-MHC class I (pMHC-I) remains incomplete. Using DNA origami nanomaterials, we precisely organize pMHC-I molecules into defined spatial configurations to systematically investigate the roles of valencies, inter-ligand spacings, geometric patterns, and molecular flexibility in regulating T cell function. We find that reducing the inter-ligand spacing to similar to 7.5 nm enhances T cell activation by up to eightfold compared to a wider spacing (similar to 22.5 nm), and that as few as six pMHC-I molecules are sufficient to elicit a robust response. Notably, the geometry of pMHC-I presentation emerges as a key determinant of signaling strength, with hexagonal arrangements proving most effective. In contrast, the introduction of flexible linkers into pMHC-I impairs TCR triggering. Together, these findings define spatial parameters that govern pMHC-I-TCR interactions at the T cell interface and provide design principles for engineering next-generation T cell-based immunotherapies.