The survival of most patients with acute myelogenous leukemia (AML) remains poor, and novel therapeutic approaches are needed to improve outcomes. Given that the fraction of AML with mutated p53 is small (∼ 10%), it appears rational to study MDM2 inhibitors as therapy for AML. Here, we report results of a detailed characterization of sensitivity and resistance to treatment ex vivo with the MDM2 inhibitor MI219 in AML blasts from 109 patients. In line with previous observations, all AML cases with mutated p53 were resistant to MI219. Importantly, approximately 30% of AML cases with unmutated p53 also demonstrated primary resistance to MI219. Analysis of potential mechanisms associated with MI219 resistance in AML blasts with wild-type p53 uncovered distinct molecular defects, including low or absent p53 protein induction after MDM2 inhibitor treatment or external irradiation. Furthermore, a separate subset of resistant blasts displayed robust p53 protein induction after MI219 treatment, indicative of defective p53 protein function or defects in the apoptotic p53 network. Finally, analysis of very sensitive AML cases uncovered a strong and significant association with mutated Flt3 status (Flt3-ITD), which for the first time identified a clinically high-risk group of AML that may particularly benefit from MDM2 inhibitor treatment. © 2010 by The American Society of Hematology.