Recently, the research of recombinant thrombopoietin (TPO) and its subsequent use in treating thrombocytopenia following radiation therapy and chemotherapy have become more important in clinics. Our study was to determine the feasibility of recombinant adeno-associated virus (rAAV)-mediated TPO gene transfer into bone marrow-derived mesenchymal stem cells (MSCs) and to evaluate the conditioned medium (CM) obtained from TPO-transduced human (h) hMSCs for promoting the process of megakaryocytopoiesis. We constructed recombinant adeno-associated viruses expressing TPO successfully, and TPO mRNA and protein were both strongly expressed in TPO-transduced hMSCs. There was no decrease in green fluorescent protein (GFP) fluorescence expression of the transduced cells with continuous passaged culturing in vitro. The CM of TPO-transduced hMSCs has been shown to enhance the number of CD41(+) cells and megakaryocytic progenitors (colony-forming unit-megakaryocyte) significantly as compared to the nontransduced control. In this study, a novel safe and efficient method of promoting the megakaryocytopoiesis was established following the TPO-transduced hMSCs. These results provide a basis for the future studies on hematopoietic regulation by hMSCs transfected with TPO.