Cytotoxic T lymphocytes (CTLs) play a crucial role in tumor killing and pathogen clearance, encompassing diverse subsets such as CD4+, NK, and γδ T cells. However, definitive biomarkers for CTLs remain elusive, as cytotoxicity-molecule expression does not always correlate with cytotoxic capacity. CTLs differentiation involves transcriptional regulation by factors like T-bet and Blimp-1, but epigenetic regulation is less clear. CTLs promote tumor killing through cytotoxic granules and death receptor pathways but may also stimulate tumorigenesis in some contexts. Enhancing CTL cytotoxicity is critical, and this review summarizes current knowledge on CTL subsets, biomarkers, differentiation mechanisms, cancer-related functions, and strategies for improving cytotoxicity. Key outstanding questions include refining CTL definitions, characterizing subtype diversity, elucidating differentiation and senescence pathways, and enabling multi-omics profiling. A comprehensive understanding of CTL biology will facilitate optimization of their immunotherapy applications. The review highlights the heterogeneity, regulation, functional roles, and enhancement strategies of CTLs in antitumor immunity, emphasizing gaps in our knowledge of subtype diversity, definitive biomarkers, epigenetic control, microbial interactions, and multi-omics characterization. Addressing these questions will refine our understanding of CTL immunology to better leverage cytotoxic functions against cancer.Cytotoxic T lymphocytes (CTLs) play a crucial role in tumor killing and pathogen clearance, encompassing diverse subsets such as CD4+, NK, and γδ T cells. However, definitive biomarkers for CTLs remain elusive, as cytotoxicity-molecule expression does not always correlate with cytotoxic capacity. CTLs differentiation involves transcriptional regulation by factors like T-bet and Blimp-1, but epigenetic regulation is less clear. CTLs promote tumor killing through cytotoxic granules and death receptor pathways but may also stimulate tumorigenesis in some contexts. Enhancing CTL cytotoxicity is critical, and this review summarizes current knowledge on CTL subsets, biomarkers, differentiation mechanisms, cancer-related functions, and strategies for improving cytotoxicity. Key outstanding questions include refining CTL definitions, characterizing subtype diversity, elucidating differentiation and senescence pathways, and enabling multi-omics profiling. A comprehensive understanding of CTL biology will facilitate optimization of their immunotherapy applications. The review highlights the heterogeneity, regulation, functional roles, and enhancement strategies of CTLs in antitumor immunity, emphasizing gaps in our knowledge of subtype diversity, definitive biomarkers, epigenetic control, microbial interactions, and multi-omics characterization. Addressing these questions will refine our understanding of CTL immunology to better leverage cytotoxic functions against cancer.