The third-generation mutation-selective EGFR tyrosine kinase inhibitor (EGFR-TKI) osimertinib (or AZD9291) effectively induces apoptosis in EGFR mutant (EGFRm) non-small cell lung cancer (NCSLC) cells. However, the underlying mechanisms have not been fully elucidated. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or TNFSF10) is known as a death ligand that initiates apoptosis via binding to its cell surface death receptors such as DR5. In this study, we found that osimertinib and other EGFR-TKIs increased the expression of TRAIL primarily in EGFRm NSCLC cell lines. This effect was accompanied with increased IL6 expression and STAT3 activation. Inhibition of STAT3 with either protein degradation or gene knockout abrogated the ability of osimertinib or recombinant human IL6 to elevate TRAIL levels. Moreover, osimertinib increased STAT3-dependent transcription of TRAIL via two STAT3 novel binding sites present in the TRAIL 5'flanking region. Hence, osimertinib induces IL6/STAT3-mediated TRAIL expression in EGFRm NSCLC cells. While osimertinib lost the ability to induce TRAIL expression in osimertinib-resistant EGFRm NSCLC, knockdown or knockout of TRAIL in sensitive EGFRm NSCLC cells rendered them less sensitive to osimertinib both in vitro and in vivo. Thus, TRAIL elevation contributes to the induction of apoptosis by osimertinib in EGFRm NSCLC cells. Furthermore, osimertinib increased membrane-bound TRAIL and DR5 membrane clustering and DR5 knockdown significantly compromised the cell-killing effect of osimertinib, together suggesting a DR5-dependent effect. Collectively, this study has revealed a previously undiscovered connection between TRAIL induction and osimertinib-induced apoptosis in EGFRm NSCLC cells, increasing our understanding of mechanisms accounting for apoptosis induced by osimertinib.