Pluripotent stem cells (PSCs) have been traditionally expanded on a two-dimensional (2D) surface and require substrates coated with extracellular matrix (ECM) proteins. Recently, PSCs have been successfully expanded in suspension as undifferentiated PSC aggregates, which offer a means for large-scale production. Toward lineage-specific differentiation, PSCs can form aggregate-like structures known as embryoid bodies (EBs). The morphology and size of EBs have been shown to significantly affect the differentiation into specific lineages and three-dimensional (3D) tissue development, thus efforts have been devoted to form size-controlled EBs. The integration of both PSC expansion and differentiation in suspension promotes PSC-derived cell production in bioreactors. However, the cellular organization and differentiation potential of PSC aggregates, as well as the role of the cues provided by the reactors to regulate EB fate, have yet to be fully understood. Despite these challenges, integrated PSC aggregate-based culture provides a platform for a simple, scalable bioprocess for the potential application of PSCs in regenerative medicine, disease modeling, and drug discovery. (C) 2016 Elsevier Ltd. All rights reserved.