The serine/threonine kinases, Akt1/PKBa, Akt2/PKBb, Akt3/PKBg, 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 MEF cells (MEF-Akt1,2-DKO). Our data indicate that Akt3 siRNA inhibits Akt3 protein expression in a dose-dependent manner. As a result, phosphorylation of Akt and its down-stream targets, including FKHRL1 and GSK3a/b, are reduced accordingly. The treatment also induces apoptosis in MEF-Akt1,2-DKO cells. However, apoptosis induction is significant only when more than 80% of Akt3 protein was depleted. Re-introducing Akt3 totally rescued Akt3-siRNA induced apoptosis in MEF-Akt1,2-DKO cells. In addition, re-introducing Akt1 also inhibited apoptosis induced by Akt3 siRNA. Moreover, Akt3 siRNA potentiates different stress-induced apoptosis in MEF-Akt1,2-DKO cells at a lower dose when compared to 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.