PTEN gene inactivation by mutation or deletion is common in pediatric T-cell acute lymphoblastic leukemia (T-ALL), but the impact on outcome is unclear, particularly in patients with NOTCH1/FBXW7 mutations. We screened samples from 145 patients treated on the MRC UKALL2003 trial for PTEN mutations using heteroduplex analysis and gene deletions using single nucleotide polymorphism arrays, and related genotype to response to therapy and long-term outcome. PTEN loss-of-function mutations/gene deletions were detected in 22% (PTEN(ABN)). Quantification of mutant level indicated that 67% of mutated cases harbored more than one mutant, with up to four mutants detected, consistent with the presence of multiple leukemic sub-clones. Overall, 41% of PTEN(ABN) cases were considered to have biallelic abnormalities (mutation and/or deletion) with complete loss of PTEN in a proportion of cells. In addition, 9% of cases had N- or K-RAS mutations. Neither PTEN nor RAS genotype significantly impacted on response to therapy or long-term outcome, irrespective of mutant level, and there was no evidence that they changed the highly favorable outcome of patients with double NOTCH1/FBXW7 mutations. These results indicate that, for pediatric patients treated according to current protocols, routine screening for PTEN or RAS abnormalities at diagnosis is not warranted to further refine risk stratification.