Intensified and central nervous system (CNS)–directed chemotherapy has improved outcomes for pediatric B cell acute lymphoblastic leukemia (B-ALL) but confers treatment-related morbidities. Moreover, many patients suffer relapses, underscoring the need to develop new molecular targeted B-ALL therapies. Using a mouse model, we show that leukemic B cells require pre–B cell receptor (pre-BCR)–independent spleen tyrosine kinase (SYK) signaling in vivo for survival and proliferation. In diagnostic samples from human pediatric and adult B-ALL patients, SYK and downstream targets were phosphorylated regardless of pre-BCR expression or genetic subtype. Two small-molecule SYK inhibitors, fostamatinib and BAY61-3606, attenuated the growth of 69 B-ALL samples in vitro, including high-risk (HR) subtypes. Orally administered fostamatinib reduced heavy disease burden after xenotransplantation of HR B-ALL samples into immunodeficient mice and decreased leukemia dissemination into spleen, liver, kidneys, and the CNS of recipient mice. Thus, SYK activation sustains the growth of multiple HR B-ALL subtypes, suggesting that SYK inhibitors may improve outcomes for HR and relapsed B-ALL.