Abstract Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of non-small cell lung cancer (NSCLC), but the predictive value of PD-L1 expression alone remains limited. Thus, there is a need for additional predictive biomarkers and functional assays to guide ICI use more precisely. Recent clinical observations suggest that NSCLC patients harboring MED12 mutations tend to respond more favorably to ICI treatment. Based on prior studies reporting that MED12 mutations can lead to functional loss of the MED12 protein, we generated MED12 knock-out NSCLC cell lines using CRISPR-Cas9 and observed significantly increased PD-L1 expression compared to control cells, as confirmed by western blotting and flow cytometry. To evaluate functional sensitivity to ICI, we developed a novel in vitro co-culture system using CAR-engineered Jurkat T cells. In this system, MED12-deficient NSCLC cells suppressed Jurkat T cell activation, which was restored upon treatment with atezolizumab, an anti–PD-L1 antibody. These results demonstrate that PD-L1 upregulation caused by MED12 loss leads to T cell inhibition, which can be reversed with PD-L1 blockade. In addition, we found that the regulation of PD-L1 expression is associated with altered YAP activity following the loss of MED12 function. This finding suggests that the YAP signaling pathway may serve as a key mediator of immune microenvironment changes in MED12-mutated tumors. In conclusion, our study is the first to demonstrate that MED12 mutation promotes an immunosuppressive environment through PD-L1 upregulation and alters ICI responsiveness in NSCLC. MED12 thus emerges as a promising predictive biomarker for ICI treatment outcomes. Furthermore, our CAR–Jurkat T cell–based in vitro assay offers a practical and quantitative platform for preclinical screening of ICI efficacy, with potential for broad application in immuno-oncology research and drug development. Citation Format: Hyun-Min Ryu, Deokhoon Kim, Shinkyo Yoon, Yunkyung Sung, Ho-Su Lee, Ji Eun Park, Chang Hoon Lee, Wanlim Kim, Seyoung Seo, Sang-We Kim, Kang-Seo Park, Dae Ho Lee. MED12 loss promotes PD-L1–mediated immune evasion and predicts response to immune checkpoint inhibitors in NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C062.