Acute lymphoblastic leukemia (ALL) dissemination to the central nervous system (CNS) is a challenging clinical problem whose underlying mechanisms are poorly understood. Here, we show that primary human ALL samples injected into the femora of immunodeficient mice migrate to the skull and vertebral bone marrow and provoke bone lesions that enable passage into the subarachnoid space. Treatment of leukemia xenografted mice with a biologic antagonist of receptor activator of nuclear factor κB ligand (RANKL) blocks this entry route. In addition to erosion of cranial and vertebral bone, samples from individuals with B-ALL also penetrate the blood-cerebrospinal fluid barrier of recipient mice. Co-administration of C-X-C chemokine receptor 4 (CXCR4) and RANKL antagonists attenuate both identified routes of entry. Our findings suggest that targeted RANKL and CXCR4 pathway inhibitors could attenuate routes of leukemia blast CNS invasion and provide benefit for B-ALL-affected individuals.