Busulfan (Bu) is used as a myeloablative agent in conditioning regimens before allogeneic hematopoietic cell transplantation (allo-HCT). In line with strategies explored in adults, patient outcomes may be optimized by replacing cyclophosphamide (Cy) with or without melphalan (Mel) with fludarabine (Flu). We compared outcomes in 2 consecutive cohorts of HCT recipients with a nonmalignant HCT indication, a myeloid malignancy, or a lymphoid malignancy with a contraindication for total body irradiation (TBI). Between 2009 and 2012, 64 children received Flu+Bu at a target dose of 80-95mg·h/L, and between 2005 and 2008, 50children received Bu targeted to 74-80mg·h/L+Cy. In the latter group, Mel was added for patients with myeloid malignancy (n=12). Possible confounding effects of calendar time were studied in 69 patients receiving a myeloablative dose of TBI between 2005 and 2012. Estimated 2-year survival and event-free survival were 82% and 78%, respectively, in the FluBu arm and 78% and 72%, respectively, in the BuCy (Mel) arm (P=not significant). Compared with the BuCy (Mel) arm, less toxicity was noted in the FluBu arm, with lower rates of acute (noninfectious) lung injury (16% versus 36%; P= .007), veno-occlusive disease (3% versus 28%; P= .003), chronic graft-versus-host disease (9% versus 26%; P= .047), adenovirus infection (3%versus 32%; P= .001), and human herpesvirus 6 infection reactivation (21% versus 44%; P= .005). Furthermore, the median duration of neutropenia was shorter in the FluBu arm (11days versus 22days; P<.001), and the patients in this arm required fewer transfusions. Our data indicate that Flu (160mg/m2) with targeted myeloablative Bu (90mg·h/L) is less toxic than and equally effective as BuCy (Mel) in patients with similar indications for allo-HCT.