Several different mutations collaborate with the fusion proteins in core-binding factor acute myeloid leukemia (CBF-AML) to induce leukemogenesis, but their prognostic significance remains unclear. We screened 354 predominantly younger (<60 years) adults with t(8;21) (n = 199) or inv(16) (n = 155) entered into UK MRC trials for KIT, FLT3 tyrosine kinase domain (FLT3TKD), N-RAS, K-RAS and c-CBL mutations and FLT3 internal tandem duplications (FLT3ITD) and assessed the impact of relative mutant level on outcome. Overall, 28% had KIT, 6% FLT3ITD, 10% FLT3TKD, 27% RAS and 6% CBL mutations. Mutant levels for all genes/loci were highly variable. KIT mutations were associated with a higher cumulative incidence of relapse but in multivariate analysis this was only significant for cases with a higher mutant level of 25% or greater (95% confidence interval (CI) = 1.01-1.52, P = 0.04). Similarly, only FLT3ITD-HIGH was a significant adverse factor for overall survival (OS; CI = 1.27-5.39, P = 0.004). Conversely, FLT3TKD-HIGH and CBLHIGH were both favorable factors for OS (CI = 0.31-0.89, P = 0.01 and CI = 0.05-0.85, P = 0.02, respectively). KIT mutations were frequently lost at relapse, which is relevant to minimal residual disease detection and the clinical use of KIT inhibitors. These results indicate that relative mutant level should be taken into account when evaluating the impact of mutations in CBF-AML.