Glycogen synthase kinase (GSK)-3, a constitutively active serine-threonine kinase, acts as a key regulator of major signaling pathways, including the Wnt, Hedgehog, and Notch pathways. Although a number of studies have demonstrated that GSK-3 plays a critical role in several cellular processes, such as differentiation, growth, and apoptosis, the effects of GSK-3 on platelet production have not been explored. There are two GSK-3 isoforms, GSK-3α and GSK-3β. GSK-3β is more highly expressed in platelets. In the present study, primary human bone marrow cells were cultured for 12 days in megakaryocyte lineage induction (MKLI) media to induce their differentiation into megakaryocyte (MK) lineage cells, in the presence or absence (+/-) of TWS119, a GSK-3β inhibitor, during MK differentiation from stem cells and subsequent platelet production. MK maturation, MK production, and subsequent platelet production were markedly enhanced in cells cultured in TWS119 (+) compared with cells cultured in TWS119 (-). These effects on MK lineage cells were thrombopoietin (TPO)-dependent. We next performed the experiment focusing on the inhibitory effect of GSK-3β on platelet production. Bone marrow cell-derived CD41 (+)/CD42b (+)/propidium iodide (-) cells in the large (MK)-sized cell population (day 8), as living mature MKs, were further cultured in the MKLI media in TWS119 (+/-) for 6 days. Platelet production from mature MKs in TWS119 (+) was approximately two-fold higher than that in TWS119 (-). The mature MKs were cultured in MKLI media in TWS119, in TPO (+/-), and platelet production was markedly decreased in TPO (-). This indicated that the GSK-3β inhibition affects thrombopoiesis under these conditions with TPO. To identify the target(s) of GSK-3β inhibition during differentiation into MK lineage cells, we performed a differential gene expression study and subsequent pathway analysis of the large (MK)-sized CD41 (+)/propidium iodide (-) cells cultured in TWS119 (+/-) for 3 days. The results of the analysis indicated that GSK-3β inhibition during differentiation into MK lineage cells affected factors related to transcription, apoptosis, cell division, cell cycle, blood coagulation, lipid transport, keratin filament, metabolic processes, and the Wnt signaling and transforming growth factor-β signaling pathways. These observations suggest that GSK-3β inhibition and TPO treatment affect both megakaryopoiesis and thrombopoiesis in an in vitro differentiation system for primary human bone marrow cells.