Abstract Addressing the formidable challenges presented by diffuse midline glioma (DMG), a notably aggressive pediatric brain tumor with limited therapeutic options, this study investigates the oncogenic roles of MYC and PDGFB and evaluates SOX9's contribution to therapeutic resistance. Utilizing advanced transgenic mouse models and the RCAS virus system, our research effectively simulates the progression and treatment responses characteristic of DMG.   In our approach, we established a DMG tumor model by manipulating Tumor Virus A receptor (TVA)-expressing embryonic neural stem cells sourced from the E12.5 hindbrain in vitro. Validation of this model and its genetic perturbations was achieved through detailed Western blot analyses. Results indicate that DMG cells overexpressing MYC tended to be significantly more sensitive to chemotherapy compared to PDGFB cells that do not overexpress MYC, positioning MYC as a crucial therapeutic target. On the other hand, increased expression of SOX9 was linked to a slight increase in resistance to conventional treatment modalities, highlighting its role in promoting adaptive resistance mechanisms within the tumor microenvironment.   This research emphasizes the critical importance of merging molecular biology techniques with comprehensive in vivo modeling to elucidate DMG's pathophysiology and identify actionable therapeutic targets. Our findings offer significant insights into the molecular dynamics of DMG and suggest novel targets for therapeutic intervention that could substantially improve clinical outcomes in this challenging pediatric malignancy. Future research should aim to broaden the genetic profiling of DMG, and tailor therapeutic approaches based on specific molecular profiles to optimize treatment efficacy and precision.