The serine/threonine kinases Akt1/PKBalpha, Akt2/PKBbeta, and Akt3/PKBgamma have been implicated in preventing cells from undergoing apoptosis. Although several small molecule inhibitors of Akt have been reported to induce apoptosis in cancer cells, these inhibitors may have additional targets. In the current study, we used an Akt3 small interfering RNA (Akt3 siRNA) to analyze apoptosis induction in Akt1 and Akt2 double knock-out mouse embryonic fibroblast cells (MEF-Akt1,2-DKO). Our data indicated that Akt3 siRNA inhibited Akt3 protein expression in a dose-dependent manner. As a result, phosphorylation of Akt and its downstream targets, including FKHRL1 and GSK3alpha/beta, were reduced accordingly. The treatment also induced apoptosis in MEF-Akt1,2-DKO cells. However, apoptosis induction is significant only when more than 80% of Akt3 protein was depleted. Reintroducing Akt3 totally rescued Akt3-siRNA-induced apoptosis in MEF-Akt1,2-DKO cells. In addition, reintroducing Akt1 also inhibited apoptosis induced by Akt3 siRNA. Moreover, Akt3 siRNA potentiated different stress-induced apoptosis in MEF-Akt1,2-DKO cells at a lower dose when compared with what is required for apoptosis induction by itself. Our study suggests that only a small portion of Akt is active in wild-type MEF cells and a threshold of Akt inhibition is required to induce apoptosis by pure Akt inhibitors. In addition, our data indicate that cells under stress require more Akt for its survival.