Oncogenic mutations of the Kit receptor tyrosine kinase occur in several types of malignancy. Juxtamembrane domain mutations are common in gastrointestinal stromal tumors, whereas mutations in the kinase activation loop, most commonly D816V, are seen in systemic mastocytosis and acute myelogenous leukemia. Kit activation-loop mutants are insensitive to imatinib mesylate and have been largely resistant to targeted inhibition. We determined the sensitivities of both Kit mutant classes to the adenosine triphosphate (ATP)-based inhibitors AP23464 and AP23848. In cell lines expressing activation-loop mutants, low-nM concentrations of AP23464 inhibited phosphorylation of Kit and its downstream targets Akt and signal transducer and activator of transcription 3 (STAT3). This was associated with cell-cycle arrest and apoptosis. Wild-type Kit-and juxtamembrane-mutant-expressing cell lines required considerably higher concentrations for equivalent inhibition, suggesting a therapeutic window in which cells harboring D816V Kit could be eliminated without interfering with normal cellular function. Additionally, AP23464 did not disrupt normal hematopoietic progenitor-cell growth at concentrations that inhibited activation-loop mutants of Kit. In a murine model, AP23848 inhibited activation-loop mutant Kit phosphorylation and tumor growth. Thus, AP23464 and AP23848 potently and selectively target activation-loop mutants of Kit in vitro and in vivo and could have therapeutic potential against D816V-expressing malignancies.