Abstract: The in vitro erythrocyte production technique provides a new solution for blood transfusion therapy, ensuring the sustainability of blood supply, reducing the dependency of blood donors, and lowering the risk of disease transmission. This review summarized the latest developments in erythrocyte production techniques in vitro, including the production of red blood cells from different cell sources. It emphasizes the cultivation techniques and the development of immortalized erythrocyte cell lines, highlighting the significance of enhancing enucleation rates and ensuring the safety of these lines. Furthermore, this review addresses the potential and challenges associated with clinical application, providing new insights for the sustainable supply and clinical practice of red blood cells in the future.