Mutations in nucleophosmin NPM1 are the most frequent acquired molecular abnormalities in acute myeloid leukemia (AML). We determined the NPM1 mutation status in a clinically and molecularly well-characterized patient cohort of 275 patients with newly diagnosed AML by denaturing high-performance liquid chromatography (dHPLC). We show that NPM1 mutations are significantly underrepresented in patients younger than 35 years. NPM1 mutations positively correlate with AML with high white blood cell counts, normal karyotypes, and fms-like tyrosine kinase-3 gene (FLT3) internal tandem duplication (ITD) mutations. NPM1 mutations associate inversely with the occurrence of CCAAT/enhancer-binding protein-alpha (CEBPA) and NRAS mutations. With respect to gene expression profiling, we show that AML cases with an NPM1 mutation cluster in specific subtypes of AML with previously established gene expression signatures, are highly associated with a homeobox gene-specific expression signature, and can be predicted with high accuracy. We demonstrate that patients with intermediate cytogenetic risk AML without FLT3 ITD mutations but with NPM1 mutations have a significantly better overall survival (OS) and event-free survival (EFS) than those without NPM1 mutations. Finally, in multivariable analysis NPM1 mutations express independent favorable prognostic value with regard to OS, EFS, and disease-free survival (DFS).