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Summary Mutation of the Gap Junction Beta 2 gene (GJB2) encoding connexin 26 (CX26) is the most frequent cause of hereditary deafness worldwide and accounts for up to 50% of non-syndromic sensorineural hearing loss cases in some populations. Therefore, cochlear CX26-gap junction plaque (GJP)-forming cells such as cochlear supporting cells are thought to be the most important therapeutic target for the treatment of hereditary deafness. The differentiation of pluripotent stem cells into cochlear CX26-GJP-forming cells has not been reported. Here, we detail the development of a novel strategy to differentiate induced pluripotent stem cells into functional CX26-GJP-forming cells that exhibit spontaneous ATP- and hemichannel-mediated Ca2+ transients typical of the developing cochlea. Furthermore, these cells from CX26-deficient mice recapitulated the drastic disruption of GJPs, the primary pathology of GJB2-related hearing loss. These in vitro models should be useful for establishing inner-ear cell therapies and drug screening that target GJB2-related hearing loss., Graphical Abstract, Highlights • Mutation in GJB2 (CX26) is the most frequent cause of hereditary deafness worldwide • Functional CX26-gap junction plaque (GJP)-forming cells were generated from iPSCs • These cells exhibited spontaneous Ca2+ transients typical of the developing cochlea • The drastic disruption of GJP was observed in in vitro disease model of GJB2 mutation, Kamiya and colleagues generated functional CX26-gap junction plaque-forming cells from iPSCs. These cells exhibited spontaneous ATP- and hemichannel-mediated Ca2+ transients typical of the developing cochlea. Furthermore, an in vitro disease model from CX26-deficient deafness mice recapitulated the drastic disruption of gap junction plaques reported to be the primary pathology of GJB2-related hearing loss.