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Background The optimal timing of initiating granulocyte-colony stimulating factor following chemotherapy in pediatric patients has not been clearly defined. This study aimed to compare the administration of granulocyte-colony stimulating factor on day 1 versus day 3 postchemotherapy in pediatric patients with Ewing sarcoma. Method A retrospective study of pediatric patients with Ewing sarcoma who received granulocyte-colony stimulating factor following chemotherapy between January 2016 and September 2018 at a comprehensive cancer center. The institution’s chemotherapy protocol for Ewing sarcoma was modified in April 2017 to include granulocyte-colony stimulating factor initiation on day 3 instead of day 1 post-chemotherapy. Febrile neutropenia requiring hospitalization, duration of hospital stay, and chemotherapy delay were compared for patients before and after the protocol change. Results Over the study period, 250 cycles were evaluated with day 1 granulocyte-colony stimulating factor and 221 cycles with day 3 granulocyte-colony stimulating factor. There were no differences between the day 1 and day 3 groups in the number of cycles associated with Febrile neutropenia requiring hospitalization (34 vs. 19, p = 0.086), and the length of Febrile neutropenia-related hospitalization (mean 4 ± 2.1 vs. 4.6 ± 1.8, p = 0.123). However, delay in chemotherapy due to neutropenia was reported in significantly more cycles in the day 1 group, compared to the day 3 group (37 vs. 16, p = 0.01). Conclusions Febrile neutropenia resulting in hospital admission and the length of hospital stay was not different between pediatric patients with Ewing sarcoma who received granulocyte-colony stimulating factor on day 1 or day 3 post-chemotherapy. Chemotherapy delay due to neutropenia was higher in patients who received granulocyte-colony stimulating factor on day 1. Larger studies are required to fully determine the impact of delayed initiation of granulocyte-colony stimulating factor.