Cytotoxic T lymphocytes (CTLs) are critical in tumor immunity, encompassing diverse subsets like CD8+, CD4+, γδ, and iNK-CTLs. Despite their role in tumor killing, defining definitive biomarkers for CTLs remains challenging. CTLs differentiate through transcriptional and epigenetic regulation, with factors like T-bet and Blimp-1 influencing their development. They kill tumor cells via cytotoxic granules and death receptor pathways, but may also promote tumorigenesis in some contexts. Enhancing CTL cytotoxicity is crucial for effective cancer immunotherapy. This review summarizes current knowledge on CTL subsets, biomarkers, differentiation mechanisms, and strategies to improve their function. Key challenges include defining CTLs, characterizing subtype diversity, understanding differentiation and senescence pathways, and elucidating CTL-microbe interactions. A comprehensive understanding of CTL biology is essential for optimizing their use in immunotherapy. The review highlights gaps in knowledge regarding biomarkers, epigenetic control, microbial interactions, and multi-omics characterization. Addressing these issues will refine our understanding of CTL immunology and improve their role in cancer treatment. CTLs play diverse roles in tumor immunity, including direct cytotoxic killing and activation of other immune cells. They can also promote tumorigenesis, highlighting the need for strategies to enhance their cytotoxic function. Current research focuses on modulating cytokine expression, reducing immune cell infiltration, and altering metabolic pathways to improve CTL function. Various approaches, including PD-1 inhibition, IL-2 modulation, and CD40L-expressing oncolytic viruses, are being explored to enhance CTL activity. These strategies aim to improve the efficacy of cancer immunotherapy by optimizing CTL function.Cytotoxic T lymphocytes (CTLs) are critical in tumor immunity, encompassing diverse subsets like CD8+, CD4+, γδ, and iNK-CTLs. Despite their role in tumor killing, defining definitive biomarkers for CTLs remains challenging. CTLs differentiate through transcriptional and epigenetic regulation, with factors like T-bet and Blimp-1 influencing their development. They kill tumor cells via cytotoxic granules and death receptor pathways, but may also promote tumorigenesis in some contexts. Enhancing CTL cytotoxicity is crucial for effective cancer immunotherapy. This review summarizes current knowledge on CTL subsets, biomarkers, differentiation mechanisms, and strategies to improve their function. Key challenges include defining CTLs, characterizing subtype diversity, understanding differentiation and senescence pathways, and elucidating CTL-microbe interactions. A comprehensive understanding of CTL biology is essential for optimizing their use in immunotherapy. The review highlights gaps in knowledge regarding biomarkers, epigenetic control, microbial interactions, and multi-omics characterization. Addressing these issues will refine our understanding of CTL immunology and improve their role in cancer treatment. CTLs play diverse roles in tumor immunity, including direct cytotoxic killing and activation of other immune cells. They can also promote tumorigenesis, highlighting the need for strategies to enhance their cytotoxic function. Current research focuses on modulating cytokine expression, reducing immune cell infiltration, and altering metabolic pathways to improve CTL function. Various approaches, including PD-1 inhibition, IL-2 modulation, and CD40L-expressing oncolytic viruses, are being explored to enhance CTL activity. These strategies aim to improve the efficacy of cancer immunotherapy by optimizing CTL function.