CXCL10 is a cytokine that is elevated during EGFR-TKI treatment in the tumor microenvironment of lung cancer. Here, we report an original study that the impact of the CXCL10/CXCR3 pathway on EGFR-TKI resistance in EGFR-mutant lung cancer through a cytokine array analysis during in vitro coculture with tumor cells and activated PBMCs treated with EGFR-TKI, as well as the serial analysis of CXCL10 in EGFR-mutant lung cancer transgenic mice during EGFR-TKI treatment. In EGFR-mutant tumor cells cocultured with activated PBMCs, EGFR-TKI treatment increased CXCL10 in the supernatant; this activated CXCR3 in the tumor cells to induce the phosphorylation of Src and the NF-κB subunit, p65, and the expression of HIF-1α. CXCL10 siRNA treatment of EGFR-mutant tumor cells also decreased CXCL10 in the supernatant from coculturing with activated PBMCs, suggesting that the effects of CXCL10 occur via autocrine and paracrine pathways. Importantly, elevated CXCL10/CXCR3 signaling was recapitulated in a transgenic lung cancer mouse model. Our results show that increased CXCL10 levels during early EGFR-TKI treatment stimulate oncogenic signaling of persistent tumor cells to contribute to EGFR-TKI resistance via autocrine and paracrine pathways.Keywords:CXCL10; CXCR3; EGFR mutation; EGFR-TKI; HIF-1α; NF-κB; lung cancer; tyrosine kinase inhibitor.