The objectives of this preclinical study were to characterize the kinase binding affinity and selectivity of quizartinib and its active metabolite AC886 compared with those of other FLT3 inhibitors, to evaluate the antitumor effect of quizartinib on midostaurin-resistant AML cells, and to assess the impact of midostaurin resistance on FLT3 inhibitors. Here, FLT3 is linked to acute myeloid leukemia.