RAD001 is currently used as an immunosuppressant and anticancer drug. Megakaryocyte (MK) differentiation includes development from pluripotent stem cells to proliferation and differentiation toward MK formation and platelet maturation. Our preliminary assay showed that RAD001 might stimulate MK differentiation; however, the exact regulatory mechanisms needed to be elucidated. By the ex vivo assay, RAD001 induced MK differentiation in human haematopoietic stem cells, with both the stimulation of CFU-GM colony formation and CD61 surface marker expression. Then, BALB/c mice were orally administrated with or without agrylin and/or RAD001 for 15 days. The platelet count and bone marrow CFU-MK colony formation were eliminated by agrylin, but unchanged in RAD001 and RAD001 plus agrylin mice. An ex vivo assay of bone marrow-derived stem cells demonstrated that RAD001 increased the number of CFU-MK colonies. The MK count in bone section indicated the decreased effect by agrylin and then recovered by RAD001. The level of plasma thrombopoietin was also enhanced in RAD001-treated mice. The effect of RAD001 on human leukaemic K562 and HEL cells showed the growth inhibition and MK differentiation activities; including morphological observation, CD41 and CD61 expression, and platelet factor 4 secretion. In RAD001-treated HEL cells, p-STAT3 expression, STAT3 translocation, and STAT3-DNA binding activity were up-regulated. Furthermore, STAT3 siRNA decreased the p-STAT3 and CD61 expression, as well as the CD61 fluorescence intensity, indicating that STAT3 may be critical in RAD001-mediated MK differentiation. Conclusion, the present study demonstrated that RAD001 might have the capacity to induce MK differentiation through the up-regulation of STAT3 signalling.