Cytotoxic T lymphocytes (CTLs) play a key role in the cell-mediated immune response against virally-infected and tumourigenic cells, killing their targets through the release of cytolytic granules. T cell receptor (TCR) recognition of foreign peptides presented by Class I MHC molecules stimulates naïve CD8+ T cell differentiation to effector cells and triggers the cytolytic activity of effector CTLs. Signal transduction downstream of the TCR is a highly diverse and coordinated network of post-translational protein modifications that ultimately drive transcriptional, translational, metabolic and cytoskeletal changes in the cell. How cytotoxic T cells coordinate their molecular machinery in response to strong versus weak stimuli remains unclear. Utilising single-cell methods including mass cytometry and single-cell RNA-sequencing, this study demonstrates how naïve and restimulated cytotoxic T cells coordinate their responses against peptides of differing stimulation strengths.