Tissue-resident memory CD8(+) T (T-RM) cells are found at common sites of pathogen exposure, where they elicit rapid and robust protective immune responses(1,2). However, the molecular signals that control T-RM cell differentiation and homeostasis are not fully understood. Here we show that mouse T-RM precursor cells represent a unique CD8(+) T cell subset that is distinct from the precursors of circulating memory cell populations at the levels of gene expression and chromatin accessibility. Using computational and pooled in vivo RNA interference screens, we identify the transcription factor Runx3 as a key regulator of T-RM cell differentiation and homeostasis. Runx3 was required to establish T-RM cell populations in diverse tissue environments, and supported the expression of crucial tissue-residency genes while suppressing genes associated with tissue egress and recirculation. Furthermore, we show that human and mouse tumour-infiltrating lymphocytes share a core tissue-residency gene-expression signature with T-RM cells that is associated with Runx3 activity. In a mouse model of adoptive T cell therapy for melanoma, Runx3-deficient CD8(+) tumour-infiltrating lymphocytes failed to accumulate in tumours, resulting in greater rates of tumour growth and mortality. Conversely, overexpression of Runx3 enhanced tumour-specific CD8(+) T cell abundance, delayed tumour growth, and prolonged survival. In addition to establishing Runx3 as a central regulator of T-RM cell differentiation, these results provide insight into the signals that promote T cell residency in non-lymphoid sites, which could be used to enhance vaccine efficacy or adoptive cell therapy treatments that target cancer.