Mast cell leukemia (MCL) is a highly fatal malignancy characterized by devastating expansion of immature mast cells in various organs. Although considered a stem cell disease, little is known about MCL-propagating neoplastic stem cells. We here describe that leukemic stem cells (LSCs) in MCL reside within a CD34 /CD38 fraction of the clone. Whereas highly purified CD34 /CD38 cells engrafted NSG mice with fully manifesting MCL, no MCL was produced by CD34 /CD38 progenitors or the bulk of KIT /CD34 mast cells. CD34 /CD38 MCL cells invariably expressed CD13 and CD133, and often also IL-1RAP, but did not express CD25, CD26 or CLL-1. CD34 /CD38 MCL cells also displayed several surface targets, including CD33, which was homogenously expressed on MCL LSCs in all cases, and the D816V mutant form of KIT. Although CD34 /CD38 cells were resistant against single drugs, exposure to combinations of CD33-targeting and KIT-targeting drugs resulted in LSC-depletion and markedly reduced engraftment in NSG mice. Together, MCL LSCs are CD34 /CD38 cells that express distinct profiles of markers and target antigens. Characterization of MCL LSCs should facilitate their purification and should support the development of LSC-eradicating curative treatment approaches in this fatal type of leukemia.