The t(12;21) translocation, which results in the fusion of the TEL (ETV6) and AML1 (RUNX1) genes, is present in ~25% of pediatric B-precusor acute lymphoblastic leukemia (ALL) patients. Although initially thought to be a favorable prognostic indicator, the t(12;21) was associated with a similar rate of relapse as t(12;21)-negative pre-B ALL in subsequent studies. While secondary cytogenetic abnormalities are often present, their prognostic significance is unknown. The objective of this study is therefore to examine the type, frequency and prognostic impact of secondary cytogenetic abnormalites in t(12;21)-positive blasts of children with ALL. We studied retrospectively 56 patients diagnosed with t(12;21)-positive ALL at the Hospital for Sick Children between 2000–2005. The mean age at diagnosis was 4.9 years (range <1 to 12 years). Nine patients (16%) presented with a white blood cell count of greater than 50 × 109/L. Cytogenetic studies consisted of a combination of FISH, G-banding and spectral karyotyping. The most frequent secondary cytogenetic changes in patients with t(12;21)-positive ALL were: the deletion of the non-translocated TEL allele (38%), an extra copy of the TEL-AML1 fusion gene (14%). Additional numerical abnormalities were seen in approximately one-third of the cases, with the most common being a gain of chromosome 21(13%). Recurrent deletions of chromosomal regions 6q and 11q were observed (14%). A surprising degree of karyotypic complexity was noted. Three or more additional chromosome abnormalities, two abnormal clonal lines, and complex structural rearrangements involving TEL-AML1 were detected in 28%, 21%, and 9% of patients, respectively. Five children (9%) in this cohort have developed a relapse of ALL. The 5-year event-free survival (EFS) was 83±7%. The 5-year EFS for patients with a deleted second TEL allele or an extra TEL-AML1 fusion was not statistically different. Of note, 4 of the children who relapsed had high-risk features at presentation (NCI criteria). All were treated with chemotherapy protocols developed for lower risk ALL. The realization that TEL-AML1 does not always predict excellent prognosis indicates that an on-going study of patients and their survival, with detailed analysis of complex genetic changes, is necessary for a reliable assessment of the prognostic value of the t(12;21) in pediatric ALL.