The powerful suppressive capabilities of regulatory T (T) cells and their appreciable contribution to tumour progression make them attractive immunotherapeutic targets. However, their role in systemic immune homeostasis makes it important to find ways to specifically target tumour-infiltrating T cells while leaving the wider system unperturbed. It is also unknown whether therapies depleting or disrupting the function of tumour-infiltrating T cells will provide the greatest efficacy while limiting immune-related adverse events. In addition, T cells share much of their biology with conventional CD4 T cells, introducing challenges when designing targeted immunotherapies. In this Review, we discuss recent advances in differentiating tumour-infiltrating T cells from their systemic and tissue-resident counterparts and understanding how the biology of tumour-infiltrating T cells differs from conventional CD4 T cells. We also discuss how recent technological advances may enable the study of tumour-infiltrating T cells in even greater detail, helping to identify new targets for next-generation immunotherapeutic drugs.