BACKGROUND Human induced pluripotent stem cell (hiPSC) technology is a valuable tool for generating patient-specific stem cells, facilitating disease modeling, and investigating disease mechanisms. However, iPSCs carrying specific mutations may limit their clinical applications due to certain inherent characteristics. AIM To investigate the impact of MERTK mutations on hiPSCs and determine whether hiPSC-derived extracellular vesicles (EVs) influence anomalous cell junction and differentiation potential. METHODS We employed a non-integrating reprogramming technique to generate peripheral blood-derived hiPSCs with and hiPSCs without a MERTK mutation. Chromosomal karyotype analysis, flow cytometry, and immunofluorescent staining were utilized for hiPSC identification. Transcriptomics and proteomics were employed to elucidate the expression patterns associated with cell junction abnormalities and cellular differentiation potential. Additionally, EVs were isolated from the supernatant, and their RNA and protein cargos were examined to investigate the involvement of hiPSC-derived EVs in stem cell junction and differentiation. RESULTS The generated hiPSCs, both with and without a MERTK mutation, exhibited normal karyotype and expressed pluripotency markers; however, hiPSCs with a MERTK mutation demonstrated anomalous adhesion capability and differentiation potential, as confirmed by transcriptomic and proteomic profiling. Furthermore, hiPSC-derived EVs were involved in various biological processes, including cell junction and differentiation. CONCLUSION HiPSCs with a MERTK mutation displayed altered junction characteristics and aberrant differentiation potential. Furthermore, hiPSC-derived EVs played a regulatory role in various biological processes, including cell junction and differentiation.