This protocol describes the rapid and robust differentiation of cortical neurons from hiPSCs via induced expression of the neurogenin-2 (NGN2) transcription factor (Wang et al., 2017; Zhang et al., 2013). To begin, iPSCs with a stably integrated human or mouse neurogenin-2 transgene under a tetracycline-inducible promoter are exposed to doxycycline in neuronal induction medium (IM). Since iPSCs grow as colonies, they must be single-cell dissociated to a new plate before doxycycline treatment in order to provide the differentiating neurons adequate space to begin producing neuritic extensions.Once the cells have been partially differentiated on Matrigel, they are re-plated onto dishes coated with poly-L-ornithine (PLO) for neuronal maturation. After 3 days of doxycycline treatment, the cells are committed to neural differentiation, although at this time they may have only minor neuritic elongations. These neurites are generally well-preserved after dissociation and replating, but the longer neuritic projections present in cells differentiated past 3 days are often damaged during the splitting process. For this reason, differentiated neurons are optimally replated on day 3. At this time, differentiating neurons can also be cryopreserved for use at a later date, enabling curation of large, standardized stocks of partially differentiated neurons for future experiments.