The most widely accepted conditioning regimen to allogeneic hematopoietic stem cell transplantation consists of total body irradiation, especially in patients affected by acute lymphoblastic leukemia (ALL). In this retrospective study, we report our experience on hematopoietic stem cell transplantation in 44 pediatric patients with acute lymphoblastic leukemia using a non-radiation–based conditioning regimen (busulfan/cyclophosphamide). Median age at transplantation was 12.5 years (range, 4 to 14 y). 39 out of 44 patients received transplants in complete remission. At a median follow-up of 390 days, the probabilities of 3-year disease-free survival and overall survival were 50% and 68%, respectively. Disease status of hematopoietic stem cell transplantation was the only significant variable affecting the overall survival. Acute and chronic graft-versus-host disease occurred in 23 (64%) and 12(18%) patients, respectively. Relapse was significantly higher among patients transplanted in advanced disease status. The results of the study indicate that non-radiation–based preparative regimens can be used in pediatric patients with ALL. However, well-designed comparative trials are needed to better clarify the difference between radiation and non-radiation–based conditioning regimens in pediatric ALL.