Natural killer (NK) cells are lymphocytes of the innate immune system that are critical in host defense. They are best known for their ability to mediate cytotoxicity, which involves a coordinated series of events resulting in the directed secretion of lytic granules onto a target cell. This process requires the formation of an immunological synapse in NK cells. The NK cell immunological synapse involves the reorganization of the actin cytoskeleton and clustering of certain cell surface receptors in the NK cell at the interface with the target cell. The lytic NK cell immunological synapse, specialized for mediating cytotoxicity, is further distinguished by the polarization of lytic granules, which are then secreted through this region onto the target cell. These events unfold in a definitive sequence and lead to critical checkpoints that provide regulatory control at specific stages in the formation of the NK cell lytic synapse.