CD8+ cytotoxic T lymphocytes (CTL) are essential for effective immune defence against intracellular microbes and neoplasia. CTL recognize short peptide fragments presented in association with major histocompatibility complex class I (MHCI) molecules on the surface of infected or dysregulated cells. Antigen recognition involves the binding of both T cell receptor (TCR) and CD8 co-receptor to a single ligand (pMHCI). The TCR/pMHCI interaction confers antigen specificity, whereas the pMHCI/CD8 interaction mediates enhanced sensitivity to antigen. Striking biophysical differences exist between the TCR/pMHCI and pMHCI/CD8 interactions; indeed, the pMHCI/CD8 interaction can be >100-fold weaker than the cognate TCR/pMHCI interaction. Here, we show that increasing the strength of the pMHCI/CD8 interaction by ~15-fold results in non-specific, cognate antigen-independent pMHCI tetramer binding at the cell surface. Furthermore, pMHCI molecules with super-enhanced affinity for CD8 activate CTL in the absence of a specific TCR/pMHCI interaction to elicit a full range of effector functions, including cytokine/chemokine release, degranulation and proliferation. Thus, the low solution binding affinity of the pMHCI/CD8 interaction is essential for the maintenance of CTL antigen specificity.