We found that <i>Mef2c<sup>S222A/S222A</sup></i> knock-in mutant mice engineered to block MEF2C phosphorylation exhibited normal hematopoiesis, but were resistant to leukemogenesis induced by <i>MLL-AF9</i> MEF2C phosphorylation was required for leukemia stem cell maintenance and induced by MARK kinases in cells.