The prognostic value of the ectopic activation of TLX3 gene expression, a major oncogenetic event associated with pediatric T-cell acute lymphoblastic leukemia, is controversial. Likewise, the frequency and the prognostic significance in pediatric T-cell acute lymphoblastic leukemia of the newly characterized NUP214-ABL1 fusion transcript is not yet clear. Two hundred children with T-cell acute lymphoblastic leukemia were treated in the French FRALLE-93 study from 1993 to 1999. The expression of TLX3, TLX1 and SILTAL1 genes was analyzed in samples from 92 patients by real-time quantitative reverse transcriptase polymerase chain reaction. Most of these samples were further studied for NUP214-ABL1 and CALM-AF10 fusion transcripts. The median follow-up was 7.9 years. At 5 years the overall survival (+/- standard deviation, %) was 62 (+/-3%) and leukemia-free survival was 58 (+/-3%). Patients with T-cell acute lymphoblastic leukemia positive for TLX3 had a poorer survival compared to those with T-ALL negative for TLX3 (overall survival: 45+/-11% vs. 57+/-5%, p=0.049). In multivariate analysis, TLX3 expression was an independent adverse risk factor predicting relapse with a hazard ratio of 2.44 (p=0.017) and an overall survival with a hazard ratio of 3.7 (p=0.001). NUP214-ABL1 was expressed in 16.6% of patients with TLX3-positive T-ALL (3 of 18); all of the patients with this association died before completion of the treatment. SILTAL expression did not significantly affect the prognosis of patients with T-cell acute lymphoblastic leukemia. Only three of 92 patients expressed the TLX1 gene and all three are alive. TLX3 gene expression is an independent risk factor predicting poor survival in childhood T-cell acute lymphoblastic leukemia. When co-expressed with TLX3, NUP214-ABL1 transcripts may increase the risk of poor survival.