Abstract The notoriously drug resistant non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths and the ABCG2 multidrug transporter was shown to be involved in NSCLC drug resistance. Direct drug interactions with this transporter have been extensively studied, while a potential modulation of ABCG2 expression by drugs or hormones is less explored. To study the drug-dependent regulation of ABCG2 expression, we used CRISPR-Cas9 and homologous recombination-mediated DNA repair to generate fluorescent lung cancer reporter cells with preserved endogenous regulatory regions. In A549 lung adenocarcinoma cells, which have two wild type copies of the ABCG2 gene, we replaced the initial coding region of ABCG2 with a green fluorescent protein (eGFP)-coding sequence. In addition, since our previous studies demonstrated that an N-terminally eGFP-tagged ABCG2 protein is properly localized and functional, we have generated A549 cells in which the native ABCG2 coding region was modified to express an eGFP-tagged ABCG2 protein. The eGFP knock-in cells sensitively reported ABCG2 transcriptional regulation, while the cells expressing an eGFP-ABCG2 fusion protein allowed the assessment of the full, functional transporter. Using the engineered A549 cells, we found that several drugs, including anti-cancer medications, glucocorticoids, HDAC inhibitors and hypoxia-mimicking agents, caused a significant induction of ABCG2 expression. In these experiments, an intronic regulatory element was found to play a key role in the specific glucocorticoid induction of ABCG2. We suggest that our transgenic reporter cell lines, amenable to high-throughput studies, can be used to investigate the induction of ABCG2-based drug resistance in cancer and predict potential drug-drug interactions. Understanding how the generation of drug-tolerant persister cells occurs via ABCG2 expression may help the selective elimination of these multidrug resistant cancer cells. Supported by the NKFIH-OTKA grant K115375. Citation Format: Daniella Kovacsics, Nikolett Mészáros, Borbála Tihanyi, Zsolt Matula, Adrienn Borsy, Edit Szabó, György Várady, Tamás Orbán, Ágota Apáti, Ábel Fóthi, Anna Brózik, Balázs Sarkadi. Pharmacological regulation of the ABCG2 multidrug transporter in A549 non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3027.