The interaction of thrombopoietin (TPO) with its receptor c-Mpl initiates intracellular signals that are critical for megakaryopoiesis. Previously we and others have shown that TPO activates PI3K and Akt and that this pathway is important for megakaryocyte growth. Here, we investigate the importance of the Akt substrate glycogen synthase kinase (GSK)-3beta in TPO signaling. GSK-3beta is phosphorylated and inhibited by Akt as part of the PI3K pathway. GSK-3beta can also be inhibited by Wnt signaling through a distinct mechanism, leading to reduced phosphorylation and accumulation of the transcription factor beta-catenin. Therefore, we asked if TPO and Wnt3a can both inhibit GSK-3beta in megakaryocytic cells, and if they can act synergistically to promote cell growth. Although both TPO and specific chemical inhibitors of GSK-3beta result in increased survival and proliferation in a megakaryocytic cell line model, treatment with Wnt3a failed to increase cell growth either in the absence or presence of TPO, despite inducing high levels of beta-catenin. Similarly, expression of a constitutively active version of beta-catenin did not increase cell growth either in the absence or presence of TPO, suggesting that the effects of GSK-3beta inhibition downstream of TPO signaling are distinct from those induced by Wnt3a and independent of beta-catenin. The growth promoting effects of TPO are not mediated by either of the two known GSK-3beta targets, cyclin D or HIF-1alpha. We conclude that GSK-3beta is phosphorylated and inhibited by TPO-induced Akt, promoting survival and proliferation in megakaryocytic cells through a pathway that does not involve beta-catenin.