The signal transducer and activator of transcription 3 (Stat3), a member of the Stat family of proteins, is commonly activated by thrombopoietic cytokines including thrombopoietin (TPO), interleukin (IL)-6, and interleukin-11. This finding strongly suggested that Stat3 has an important role in megakaryopoiesis and thrombopoiesis. To clarify the functional role of Stat3 in in vivo megakaryopoiesis and thrombopoiesis, we generated transgenic mice overexpressing a dominant-negative Stat3, Stat3F, to suppress the function of endogenous Stat3. To accomplish the selective expression of Stat3F in megakaryocytic lineage cells, we used the regulatory gene region of GATA-1 transcription factor selectively expressed in megakaryocytic and erythroid lineage cells. Two independent transgenic (Tg) mice lines were established. It was confirmed by Western blotting analysis that Stat3F proteins were highly expressed in the platelets from the Tg mice. In addition, it was found that Stat3 activation induced by TPO stimulation was drastically suppressed in these Tg mice compared with littermates. These findings indicate that Stat3F works well in the Tg mice. Platelet counts were within the normal range in steady-state conditions and were recovered normally from transient thrombocytopenia induced by antiplatelet serum injection. Interestingly, the platelet recovery from myelosuppression after 5-fluorouracil treatment was significantly delayed in the Tg mice. Collectively, our results strongly suggest that Stat3 plays an important role in the early stage of megakaryopoiesis, presumably through the expansion of megakaryocytic progenitor cells.