Many types of immune cells rely on secretory lysosomes to execute their function. These specialised organelles perform the catabolic function of conventional lysosomes and possess the added capacity to undergo regulated exocytosis. In particular, the cytotoxic function of natural killer (NK) cells depends on the rapid, polarised secretion of secretory lysosomes, known as lytic granules, for the targeted destruction of malignant and virally infected cells. Dysregulation of the biogenesis and exocytosis of secretory lysosomes is the cause of several severe immunological diseases such as Chédiak-Higashi syndrome (CHS). In CHS, mutations in the LYST gene result in enlarged secretory lysosomes that cannot undergo exocytosis. However, the mechanisms underlying LYST function remain elusive. Here, characterisation of the giant lysosome phenotype of beige cells reveals altered phosphoinositide metabolism and increased autophagy, indicating a potential role for LYST in regulating these pathways. Furthermore, RNA interference of LYST recapitulated the giant lysosome phenotype, suggesting a strategy for analysis of the pathways in which LYST functions. Pharmacological inhibition of the phosphoinositide kinase PIKfyve induces a giant vesicle phenotype resembling that in beige cells. However, on further analysis PIKfyve inhibition was shown to induce giant endosomes as opposed to the giant lysosomes found in beige cells. Despite these differences, PIKfyve inhibition, like LYST mutations, reduced NK cell granule exocytosis at a late stage in the pathway. Thus, LYST and PIKfyve act upon different intracellular compartments but inhibit the formation and exocytosis of NK cell granules. These results extend our knowledge of the exocytosis of secretory lysosomes within the immune system and suggest future strategies to define the pathways by which lysosomal biogenesis and exocytosis is regulated.