The TLR family plays a fundamental function in the recognition of pathogens and activation of innate immunity. TLR2 is a membrane protein that is expressed on the surface of certain cells and recognizes foreign substances; it initiates a cascade of innate immune responses. TLR2 recognizes many bacterial, fungal, and viral components, as well as certain endogenous substances. We generated biallelic mutants of the TLR2 gene in human induced pluripotent stem cells (hiPSCs) using a CRISPR-Cas9 genome editing method. The TLR2 heterozygous-knockout hiPSCs retained normal morphology, gene expression, and in vivo differentiation potential.