Nucleophosmin (NPM1)–mutated acute myeloid leukemia (AML), which is recognized as a provisional entity in the World Health Organization 2008 classification of myeloid neoplasms, accounts for 30% of AML. We analyzed 1227 diagnostic and follow-up samples in 252 NPM1-mutated AML patients with 17 different NPM1 mutation–specific real-time quantitative polymerase chain reaction (RQ-PCR) assays. Paired diagnostic/relapse samples of 84 patients revealed stable NPM1 mutations in all cases, suggesting that they are pathogenetically early events and thus applicable for minimal residual disease detection. A total of 47 relapses were predictable because of an NPM1 mutation level (%NPM1/ABL1) increase of at least 1 log or in 15 cases because of NPM1 mutation levels not decreasing less than 3 log ranges. A high prognostic value of NPM1 levels was shown for 4 different intervals after therapy was initiated. Furthermore, thresholds of 0.1 and 0.01%NPM1/ABL1 during/after treatment discriminated between prognostic subgroups. Univariate analyses, including age, white blood cell count, blast count, CD34 positivity, FLT3 mutations status, FAB type, karyotype, NPM1 mutation type, and pretreatment NPM1 mutational level, showed that, besides NPM1 mutation level, only age and FLT3-LM mutation status were prognostically significant for EFS. Multivariate analysis, including age, FLT3-LM status, and NPM1 mutation level at different time points, demonstrated that NPM1 level was the most relevant prognostic factor during first-line treatment. Similar results were obtained in patients undergoing second-line chemotherapy or allogeneic stem cell transplantation.