The FOXO1 transcription factor plays an essential role in the regulation of proliferation and survival programs at early stages of B-cell differentiation. Here, we show that tightly regulated FOXO1 activity is essential for maintenance of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Genetic and pharmacological inactivation of FOXO1 in BCP-ALL cell lines produced a strong antileukemic effect associated with CCND3 downregulation. Moreover, we demonstrated that CCND3 expression is critical for BCP-ALL survival and that overexpression of CCND3 protected BCP-ALL cell lines from growth arrest and apoptosis induced by FOXO1 inactivation. Most importantly, pharmacological inhibition of FOXO1 showed antileukemia activity on several primary, patient-derived, pediatric ALL xenografts with effective leukemia reduction in the hematopoietic, lymphoid, and central nervous system organ compartments, ultimately leading to prolonged survival without leukemia reoccurrence in a preclinical in vivo model of BCP-ALL. These results suggest that repression of FOXO1 might be a feasible approach for the treatment of BCP-ALL.