Glioblastoma (GBM) is the most common and deadly adult central nervous system cancer. Despite surgical resection combined with DNA-damaging radiation and chemotherapy, GBM almost invariably recurs, becoming more resistant to radiation. To investigate the drivers of this radioresistance, we conducted a knockout radiosensitization screen and identified the High Mobility Group B2 (HMGB2) protein as a potential contributor. To elucidate HMGB2's role in GBM radioresistance, we performed viability, clonogenic survival, extreme limiting dilution (ELDA), and deletion mutant assays on patient-derived glioblastoma stem cells (GSCs) treated with a combination of radiotherapy (RT) and either HMGB2 knockdown or inhibition using Inflachromene (ICM), a small molecule inhibitor of HMGB2. Without radiation, ICM showed IC-50 values ranging from 8.43 µM to 10 µM across three GSC lines. Both ICM treatment and HMGB2 knockdown significantly reduced neurosphere formation compared to cultures treated with only RT and vehicle. Knockdown of endogenous HMGB2 combined with overexpression of an Acidic Tail-deleted HMGB2 mutant produced distinct puncta under fluorescence microscopy, unlike other deletion mutants. These findings implicate HMGB2 in GBM radioresistance and suggest that the Acidic Tail region mediates chromatin binding, possibly playing a role in HMGB2's mechanism of action. Together, these results provide a foundation for clarifying HMGB2's role in GBM biology and it's potential relevance to improving therapeutic response.