The culture and ex vivo engineering of red blood cells (RBCs) can help characterize genetic variants, model diseases, and may eventually spur the development of applications in transfusion medicine. In the last decade, improvements to the in vitro production of RBCs have enabled efficient erythroid progenitor proliferation and high enucleation levels from several sources of hematopoietic stem and progenitor cells (HSPCs). Despite these advances, there remains a need for refining the terminal step of in vitro human erythropoiesis, i.e., the terminal maturation of reticulocytes into erythrocytes, so that it can occur without feeder or accessory cells and animal-derived components. Here, we describe the near-complete erythroid differentiation of cultured RBCs (cRBCs) from adult HSPCs in accessory-cell-free and xeno-free conditions. The approach improves post-enucleation cell integrity and cell survival, and it enables subsequent storage of cRBCs for up to 42 days in classical additive solution conditions without any specialized equipment. We foresee that these improvements will facilitate the characterization of RBCs derived from gene-edited HSPCs.