Erythrocytes (red blood cells, RBCs) facilitate gas exchange in the lungs and transport oxygen to the tissues. The human body must maintain erythrocyte regeneration to support metabolically active cells and tissues. In many hematological diseases, erythrocyte regeneration is impaired. Researchers have studied erythrocyte regeneration for many years both in vivo and in vitro. In this review, we summarize the sources and main culture methods for generating mature and functional red blood cells in vitro. Hematopoietic stem cells (HSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are classic sources for erythrocyte regeneration. In addition, alternative sources such as immortalized adult human erythroid cell lines and transformed fibroblasts have also been generated and have produced functional red blood cells. The culture systems for erythrocytes differ among laboratories. Researchers hope that improvements in culture techniques may contribute to improved RBC outcomes for blood transfusions, drug delivery and the treatment of hematological diseases.