Background: Glioblastoma (GBM) is one of the most common high grade gliomas in pediatric patients, accounting for 2.8% of central nervous system tumors. Standard of care for glioblastoma includes surgical resection, radiation, and temozolomide chemotherapy. However, many pediatric patients are not suitable candidates for surgery, and their tumors inevitably develop temozolomide resistance. Despite aggressive treatment, temozolomide (TMZ) resistance leads to tumor recurrence in 90% of patients. Thus, outcome for children with pediatric GBM remains poor with a 2-year survival rate ranging from 10-30%, highlighting the need for new therapeutic approaches. Connexin-43 (Cx43) and PIK3CB are proteins involved in conferring temozolomide resistance in glioblastoma. Combined inhibition of these proteins could improve temozolomide sensitivity and help develop a selective therapy that significantly improves quality of life and prognosis in pediatric glioblastoma patients. Methods: Primary GBM cells were plated in a 96-well plate and treated with vehicle (DMSO), TMZ (50 μmol/L), TGX-221 (20 μmol/L), αCT1 (30 μmol/L), or a combination of the latter three drugs. Cell viability was measured using the MTS assay. Primary pediatric GBM cell lines SJ-GBM2 and CHLA-200 were tested. These drug combinations were also tested in normal human astrocytes to make sure there were no off-target effects. Results: We found that targeting PIK3CB and Cx43 in primary pediatric GBM increases their sensitivity to TMZ. The triple combination of TMZ, TGX-221, and αCT1 significantly decreased cell viability than each drug alone while sparing normal human astrocytes. Conclusions: Our findings suggest that inhibiting PIK3CB and Cx43 enhances therapeutic response to TMZ and limits off-target effects in pediatric GBM. These findings closely mirror our results using the triple combination in adult glioblastoma as well, thus laying the groundwork for a novel combination therapeutic that can be used across age groups.