Significance Aminoacyl-tRNA synthetases (aaRSs) catalyze aminoacylation of tRNAs in the first step of protein synthesis in the cytoplasm. However, in higher eukaryotes, they acquired additional functions beyond translation. In the present study, we show that an activated form of tyrosyl-tRNA synthetase (YRS ^ACT ) functions to enhance megakaryopoiesis and platelet production in vitro and in vivo. These findings were confirmed with human megakaryocytes differentiated from peripheral blood CD34 ⁺ hematopoietic stem cells and with human induced pluripotent stem (iPS) cells. The activity of YRS ^ACT is independent of thrombopoietin (TPO), as evidenced by expansion of the megakaryocytes from iPS cell-derived hematopoietic stem cells from a patient deficient in TPO signaling. These findings demonstrate a previously unrecognized function of an aaRS which may have implications for therapeutic interventions.