Glioblastoma (GBM) is the most common primary brain malignancy in the U.S. with a 5-year overall survival < 5% despite an aggressive standard of care. Laser interstitial thermal therapy (LITT) is a surgical approach to treating GBM that has gained traction, providing a safe option for reducing intracranial tumor burden. LITT is believed to potentially modulate GBM immune responses; however, the biochemical mechanisms underlying the modulation of immune checkpoints in GBM cells have been poorly characterized. The present study aimed to preliminarily evaluate the effects of thermal therapy and radiation on PD-L1 modulation in vitro, as a function of IDH mutational status. U87 cells and their IDH-mutant counterpart (U87), which was generated using a crispr-cas9 knock-in approach, were utilized for this preliminary evaluation. Cell heating was achieved by harvesting with trypsin centrifugation where the cell pellets were treated on a heat block for the associated time and temperature. Following thermal therapy, cells were resuspended and irradiated using a 37-Cesium irradiator at 0.6 Gy min. Immediately following treatment, cells were either plated as single cells to allow colonies to form, and stained with Coomassie blue to be counted approximately 10-14 days later or harvested for Western blot analysis. Cell lysates were analyzed for PD-L1 expression with respect to various iron metabolic parameters (mortalin (HSPA9), transferrin receptor, and ferritin heavy chain) using a Western blotting approach. In both U87 and U87 cell lines, thermal therapy showed a temperature-dependent cell-killing effect, but U87 cells appeared more sensitive to thermal treatment when treated at 43 °C for 10 min. Moreover, thermal therapy had minimal effects on cell responses to 2 Gy irradiation. Treatment with thermal therapy downregulated PD-L1 expression in U87 cells, which was associated with increased expression of the mitochondrial iron metabolic enzyme, HSPA9. Thermal therapy reversed the radiation-induced overexpression of PD-L1, transferrin receptor, and ferritin heavy chain in U87 cells. No effects were observed in wild-type U87 cells. Moreover, Ga(NO) depleted mitochondrial iron content which, in turn, significantly enhanced the sensitivity of U87 cells to thermal therapy and 2 Gy irradiation and caused a significant increase in PD-L1 expression. These results suggest that thermal therapy alone can modulate the immune checkpoint PD-L1. This effect was more pronounced when thermal therapy was combined with radiation. Mechanistically, mitochondrial iron trafficking through HSPA9 may coordinate the regulation of PD-L1 in the context of thermal therapy and ionizing radiation, which can be targeted with gallium-based therapy. These novel, preliminary findings warrant further mechanistic investigations in pre-clinical models of LITT.