Phosphatidylinositol 3-kinase (PI3K) has been shown to play an important role in collagen-induced platelet activation, but the role(s) of PTEN, a major regulator of the PI3K/Akt signaling pathway, has not been examined in platelets. Here, we report that Pten^−/− mouse blood contains 25% more platelets than Pten^+/+ blood and that PTEN deficiency significantly shortened the bleeding time, increased the sensitivity of platelets to collagen-induced activation and aggregation, and enhanced phosphorylation of Akt at Ser473 in response to collagen. Furthermore, we found that PP2, and the combination of apyrase, indomethacin + 1B5, respectively, inhibited collagen-induced aggregation in both PTEN^+/+ and PTEN^−/− platelets. In contrast, LY294002 (a PI3K inhibitor) prevented the aggregation of PTEN^+/+, but not PTEN^−/−, platelets. Therefore, PTEN apparently regulates collagen-induced platelet activation through PI3K/Akt-dependent and -independent signaling pathways.