Human NK cells are characterized by their ability to initiate an immediate and direct cytolytic response to virally infected or malignantly transformed cells. Within human peripheral blood, the more mature CD56 dim NK cell efficiently kills malignant targets at rest, whereas the less mature CD56 bright NK cells cannot. In this study, we show that resting CD56 bright NK cells express significantly more phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein when compared with CD56 dim NK cells. Consistent with this, forced overexpression of PTEN in NK cells resulted in decreased cytolytic activity, and loss of PTEN in CD56 bright NK cells resulted in elevated cytolytic activity. Comparable studies in mice showed PTEN overexpression did not alter NK cell development or NK cell–activating and inhibitory receptor expression yet, as in humans, did decrease expression of downstream NK activation targets MAPK and AKT during early cytolysis of tumor target cells. Confocal microscopy revealed that PTEN overexpression disrupts the NK cell’s ability to organize immunological synapse components including decreases in actin accumulation, polarization of the microtubule organizing center, and the convergence of cytolytic granules. In summary, our data suggest that PTEN normally works to limit the NK cell’s PI3K/AKT and MAPK pathway activation and the consequent mobilization of cytolytic mediators toward the target cell and suggest that PTEN is among the active regulatory components prior to human NK cells transitioning from the noncytolytic CD56 bright NK cell to the cytolytic CD56 dim NK cells.