Deregulation of the hematopoietic stem cell (HSC) compartment represents a hallmark of acute myeloid leukemia (AML). Recently, in vivo screening for genes that are involved in the regulation of HSCs has led to the discovery of Musashi-2 (MSI2) as a key regulator of HSCs and as a suppressor of NUMB. In order to analyze the prognostic importance of MSI2 and NUMB expression in AML, MSI2 and NUMB transcript levels from 454 AML patients treated in multicenter trials AML SHG 0199 (ClinicalTrials Identifier NCT00209833) and 0295, and 38 healthy volunteers were analyzed by reverse transcriptase PCR in the context of other molecular markers (NPM1, FLT3, CEBPA, IDH1/IDH2, DNMT3A, NRAS, WT1, KIT, MN1, BAALC, ERG, and WT1). In AML, patients with high MSI2 expression were more likely to be FLT3-ITD positive (P < .001), NPM1 (P < .001), and DNMT3A (P = .003) mutated. Overall survival (OS) was shorter in AML patients with high MSI2 expression (hazard ratio, 1.48; 95 % confidence interval, 1.13–1.95, P = .005). However, relapse-free survival (RFS, P = .15) and complete remission (CR, P = .39) rates were not influenced by MSI2 expression. In multivariate analysis, MSI2 expression remained an independent prognostic factor for OS (P = .03). NUMB expression had no impact on survival (OS, P = .47; RFS, P = .59) and CR rate (P = .39). MSI2 but not NUMB is associated with shorter OS in AML patients and may indicate a more aggressive form of AML.