The human Y chromosome harbors genes that are mainly involved in the growth, development, sexual dimorphism, and spermatogenesis process. Despite many studies, the function of the male-specific region of the Y chromosome (MSY) awaits further clarification, and a cell-based approach can help in this regard. In this study, we have developed four stable transgenic male embryonic stem cell (ESCs) lines that can overexpress male-specific genes HSFY1, RBMY1A1, RPS4Y1, and SRY. As a proof of principle, we differentiated one of these cell lines (RPS4Y1 over-expressing ESCs) to the neural stem cell (rosette structure) and characterized them based on the expression level of lineage markers. RPS4Y1 expression in the Doxycycline-treated group was significantly higher than control groups in transcription and protein levels. Furthermore, we found Doxycycline-treated group had a higher differentiation efficiency than the untreated control groups. Our results suggest that the RPS4Y1 gene may play a critical role in neurogenesis. Also, the generated transgenic ESC lines can be widely employed in basic and preclinical studies, such as sexual dimorphism of neural and cardiac functions, the development of cancerous and non-cancerous disease models, and drug screening.