Anticipated shortages in donated blood supply have prompted investigation of alternative approaches for in-vitro production of red blood cells (RBCs), such as expansion of conditional immortalization erythroid progenitors. However, there is a bioprocessing challenge wherein factors promoting maximal cell expansion and growth-limiting inhibitory factors are yet to be investigated. We used an erythroblast cell line (ImEry) derived from immortalizing CD71+CD235a+ erythroblast from adult peripheral blood for optimization of expansion culture conditions. Design of Experiments (DOE) was used in media formulation to explore relationships and interactive effects between factors which affect cell expansion. Our in-house optimized medium formulation produced significantly higher cell densities (3.62 ± 0.055) x106 cells/mL, n=3) compared to commercial formulations (2.07 ± 0.055) x106 cells/mL, n=3; at 209 hour culture). Culture media costs per unit of blood was shown to have a 2.96 - 3.09 times cost reduction. As a proof of principle for scale up, ImEry were expanded in a half-litre stirred-bioreactor under controlled settings. Growth characteristics, metabolic and molecular profile of the cells were evaluated. ImEry had identical O2 binding capacity to adult erythroblasts. Amino acid supplementation resulted in further yield improvements. Our study serves as a first step for scaling up erythroblast expansion in controlled bioreactors.