Medulloblastoma (MB) is a primary pediatric brain cancer that is frequently accompanied by metastasis and poor long-term prognosis. Our goal is to identify new signaling pathways that regulate the treatment-resistant MB cellular phenotypes, namely the stem cells. The OTX2 gene, which is amplified or overexpressed in the majority of Group 3 and 4 MBs, is a central regulator of stem cell function in these tumors. Here, we characterized the OTX2 regulatory network and identified a negative correlation between OTX2 and axon guidance genes in Group 3 and 4 MB stem/progenitor cells and tumors. Functional validation studies demonstrated that increased levels of semaphorin 4D (SEMA4D), L1 Cell Adhesion Molecule (L1CAM), and neuropilin-1 (NRP1) are associated with decreased self-renewal and that the downstream RHO signaling may be contributing to these effects. Our results offer critical insights into the molecular drivers of these aggressive tumors and provide a framework to pursue novel therapies.