The cytotoxic activity of lymphocytes is particularly dependent on the regulated and polarised delivery of lytic granules to infected or malignant cells. Although genetic and mechanistic studies have identified a number of factors that regulate exocytosis in cytotoxic lymphocytes, a systematic mapping of the relevant factors and their relationships is lacking. Here, using a genome-scale CRISPR knockout screen in human natural killer cells, we characterise a complex genetic network regulating cytotoxic granule exocytosis, with lipid metabolism and protein lipidation among the most prominent pathways. By combining global protein lipidation and membrane lipid composition studies, we further uncover the critical role of ZDHHC17 in SNAP23 palmitoylation and targeting of palmitoylated SNAP23 to the membrane for cytotoxic granule fusion and release. Using ODYA-17 labelling of Ctrl and sgZDHHC17 NK-92 cells followed by pulldown and proteomic analysis, we investigated the palmitoylome of the NK-92 model cell line. By comparing the abundance of ODYA-17 labelled proteins in the lysates of Ctrl and sgZDHHC17 NK-92 cells, we defined the molecules that depend on the activity of the palmitoyl transferase ZDHHC17.