The study of human induced pluripotent stem cells (iPSCs) and developing the technology for their practical use is one of the most knowledge-intensive areas of modern biomedical research. Despite the potential of using iPSCs in personalized medicine and to build cell-based models for disorders of various etiology, iPSC utilization remains challenging. Thus, the iPSC intercellular heterogeneity and the lack of effective identity determination and assessment methods considerably hamper reproducibility of such studies. The study was aimed to generate an iPSC line carrying the gene encoding the ATOH1 transcription factor controlled by the Tet-One expression induction system, along with TagBFP2 fluorescent protein and the puromycin resistance gene for cell selection. Molecular cloning, lentiviral transduction, cell culturing, immunofluorescence staining, and fluorescence microscopy were used during the study. The created cell model will allow analyzing the state of single cells and, therefore, has great practical potential for both laboratory and medical research.