Natural killer (NK) cells are lymphocytes of the innate immune system that participate in host defense by secreting the contents of specialized secretory organelles termed lytic granules onto virally infected or tumorigenic cells in order to terminate them. Lysis of these target cells is a highly regulated, stepwise process beginning with actin rearrangement into an immunological synapse (IS) at the contact site between NK cell and target cell, followed by microtubule organizing center (MTOC) polarization towards the target cell, and culminating in the release of lytic granule contents through the actin network at the IS. The NK cell is capable of directing lytic granules along microtubules to the IS as lytic granule contents are secreted only onto susceptible target cells while bystanding cells are protected. Through quantitative fluorescence microscopy of NK cell lines and primary NK cells, we, in fact, find that lytic granules organize around the MTOC prior to MTOC polarization to the IS. This process is dynein-dependent but independent of F-actin and microtubule reorganization. Interestingly, lytic granule convergence is a default preparatory step, found to occur even after adhesion to a non-susceptible cell. We further find that the process of lytic granule convergence is triggered both by activating receptors and soluble cytokine. In both scenarios, through the use of specific inhibitors, we demonstrate that the trigger for lytic granule convergence is Src kinase-dependent. Thus, lytic granule convergence represents a novel preparatory step in NK cell cytotoxicity, prerequisite to directed secretion. This dynein-dependent mechanism is induced very early by Src kinases after receptor and cytokine stimulation, the purpose of which may be to ensure precision in the delivery of deadly lytic granule contents.