Abstract Cellular hypoxic condition is a key event in several human cancers but the knowledge about its role in childhood ALL is very limited. In the present study gene expression profile of hypoxia-related genes (HIF1A, CA9, VEGF and SCL2A1) was evaluated in bone marrow samples of 113 pediatric patients. HIF1A mRNA up-regulation was significantly associated with higher 5-years event-free survival rates in B-ALL patients as well as in the overall ALL population in both univariate and multivariate analysis (P = 0.023 and P = 0.041 respectively). In gene expression analysis, low oxygen levels promoted HIF1A activation in a time-dependent manner, in both ALL cell lines. In vitro cytotoxic assays suggested an initial trend to hypoxia-related resistance in the first 24 hours, but later time points (48 - 72 hours) evaluation clearly showed that there is no relevant difference in drug response when comparing hypoxic to normal oxygen level conditions. Modulation of mRNA expression of several hypoxia-related genes was also observed after hypoxic exposure in a cell specific manner, suggesting that HIF1A mRNA expression could play a different role in specific subtypes of leukemia. Despite the remaining questions regarding hypoxia-mediated mechanisms, these findings could be helpful to provide new insights in the role of hypoxia in childhood ALL.