Recurrent molecular markers have been routinely used in acute myeloid leukemia (AML) for risk assessment at diagnosis, whereas their post-induction monitoring still represents a debated issue. We evaluated the prognostic value and biological impact of minimal residual disease (MRD) and of the allelic ratio (AR) of FLT3-internal-tandem duplication (ITD) in childhood AML. We retrospectively screened 494 children with de novo AML for FLT3-ITD mutation, identifying 54 harboring the mutation; 51% of them presented high ITD-AR at diagnosis and had worse event-free survival (EFS, 19.2 versus 63.5% for low ITD-AR, <0.05). Forty-one percent of children with high levels of MRD after the 1st induction course, measured by a patient-specific real-time-PCR, had worse EFS (22.2 versus 59.4% in low-MRD patients, P<0.05). Next, we correlated these parameters with gene expression, showing that patients with high ITD-AR or persistent MRD had characteristic expression profiles with deregulated genes involved in methylation and acetylation. Moreover, patients with high CyclinA1 expression presented an unfavorable EFS (20.3 versus 51.2% in low CyclinA1 group, P<0.01). Our results suggest that ITD-AR levels and molecular MRD should be considered in planning clinical management of FLT3-ITD patients. Different transcriptional activation of epigenetic and oncogenic profiles may explain variability in outcome among these patients, for whom novel therapeutic approaches are desirable.