CD8 cytotoxic T lymphocytes (CTLs) play a crucial role in anti-tumor immunity. In a previous study, we identified a subset of murine effector CTLs expressing the hepatocyte growth factor (HGF) receptor, c-Met (c-Met CTLs), that are endowed with enhanced cytolytic capacity. HGF directly inhibited the cytolytic function of c-Met CTLs, both in 2D in vitro assays and in vivo, leading to reduced T cell responses against metastatic melanoma. To further investigate the role of c-Met CTLs in a three-dimensional (3D) setting, we studied their function within B16 melanoma spheroids and examined the impact of cell-cell contact on the modulation of inhibitory checkpoint molecules' expression, such as KLRG1, PD-1, and CTLA-4. Additionally, we evaluated the cytolytic capacity of human CTL clones expressing c-Met (c-Met) and compared it to c-Met CTL clones. Our results indicated that, similar to their murine counterparts, c-Met human CTL clones exhibited increased cytolytic activity compared to c-Met CTL clones, and this enhanced function was negatively regulated by the presence of HGF. Taken together, our findings highlight the potential of targeting the HGF/c-Met pathway to modulate CTL-mediated anti-tumor immunity. This research holds promise for developing strategies to enhance the effectiveness of CTL-based immunotherapies against cancer.