Abstract: In vitro erythrocyte manufacturing technology offers a novel solution for transfusion therapy, ensuring sustainable blood supply, reducing dependence on donors, and minimizing the risk of infectious disease transmission. This review summarizes recent advancements in in vitro erythrocyte production, including techniques for generating red blood cells (RBC) from various cell sources, with a focus on the cultivation and development of immortalized erythroid cell lines. The importance of enhancing enucleation efficiency and ensuring the safety of immortalized erythroid cells is emphasized. Additionally, the potential and challenges in clinical translation are discussed, aiming to provide new insights for future sustainable RBC supply and therapeutic applications.