There is an imbalance between the supply and demand of functional red blood cells (RBCs) in clinical appli-cations. This imbalance can be addressed by regenerating RBCs using several in vitro methods. Induced pluripotent stem cells (iPSCs) can handle the low supply of cord blood and the ethical issues in embryonic stem cell research, and provide a promising strategy to eliminate immune rejection. However, no complete single-cell level differentiation pathway exists for the iPSC-derived erythroid differentiation system. In this study, we used iPSC line BC1 to establish a RBC regeneration system. The 10X Genomics single-cell transcriptome platform was used to map the cell lineage and differentiation trajectory on day 14 of the regeneration system. We observed that iPSC differentiation was not synchronized during embryoid body (EB) culture. The cells (on day 14) mainly consisted of mesodermal and various blood cells, similar to the yolk sac hematopoiesis. We identified six cell classifications and characterized the regulatory transcription factor (TF) networks and cell–cell contacts underlying the system. iPSCs undergo two transformations during the differentiation trajectory, accompanied by the dynamic expression of cell adhesion molecules and estrogen-responsive genes. We iden-tified erythroid cells at different stages, such as burst-forming unit erythroid (BFU-E) and orthochromatic erythroblast (ortho-E) cells, and found that the regulation of TFs (e.g., TFDP1 and FOXO3) is erythroid-stage specific. Immune erythroid cells were identified in our system. This study provides systematic theoretical guidance for optimizing the iPSC-derived erythroid differentiation system, and this system is a useful model for simulating in vivo hematopoietic develo-pment and differentiation.