Differentiation of O-negative Rhesus factor D negative (O-neg) human induced pluripotent stem cells (hiPSCs) can potentially generate universal donor red blood cells (RBCs) that may be useful for transfusion applications. Among the approaches described for RBCs generation, embryoid body (EB)-mediated differentiation approaches developed with xeno-free and defined conditions appear to be most feasible for future clinical development. However, conventional approaches for EB generation such as by forced aggregation have not yet been successfully demonstrated on large-scale in suspension culture. Culture of hiPSC as 3-dimensional (3D)-aggregates1 or on defined extracellular matrix (ECM)-coated microcarriers (MCs)2 are possible means for scaling up human pluripotent stem cells (hPSC) and EB expansion in suspension culture. We have previously shown that hPSC-MC aggregates could be differentiated into hematopoietic precursors3 and erythroblasts4 when differentiated with a BMP4-based protocol.4 However, repeated attempts to differentiate multiple hPSC lines initially expanded under continuous agitation condition demonstrated variability in erythroid differentiation. It has been hypothesized that agitation shear stress could induce expression of SMAD75,6 which is known to have inhibitory effects7 on phosphorylation of SMAD 1, 5 and 8, components of the TGF-β signaling pathway activated by BMP4 during the initial stages of mesodermal differentiation.8 Thus, inhibition of BMP4 signaling in agitated cultures could be a possible reason for poor mesoderm induction and variability in differentiation outcomes.