Embryoid bodies (EBs) are three-dimensional aggregates of pluripotent stem cells. EBs are differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. Background The pluripotent cell types that comprise embryoid bodies include embryonic stem cells (ESCs) derived from the blastocyst stage of embryos from mouse (mESC), primate, and human (hESC) sources. Additionally, EBs can be formed from embryonic stem cells derived through alternative techniques, including somatic cell nuclear transfer or the reprogramming of somatic cells to yield induced pluripotent stem cells (iPS). Similar to ESCs cultured in monolayer formats, ESCs within embryoid bodies undergo differentiation and cell specification along the three germ lineages – endoderm, ectoderm, and mesoderm – which comprise all somatic cell types. In contrast to monolayer cultures, however, the spheroid structures that are formed when ESCs aggregate enables the non-adherent culture of EBs in suspension, making EB cultures inherently scalable, which is useful for bioprocessing approaches, whereby large yields of cells can be produced for potential clinical applications. Additionally, although EBs largely exhibit heterogeneous patterns of differentiated cell types, ESCs are capable of responding to similar cues that direct embryonic development. Therefore, the three-dimensional structure, including the establishment of complex cell adhesions and paracrine signaling within the EB microenvironment, enables differentiation and morphogenesis which yields microtissues that are similar to native tissue structures. Such microtissues are promising to directly or indirectly repair damaged or diseased tissue in regenerative medicine applications, as well as for in vitro testing in the pharmaceutical industry and as a model of embryonic development. Formation EBs are formed by the homophilic binding of the Ca2+ dependent adhesion molecule E-cadherin, which is highly expressed on undifferentiated ESCs. When cultured as single cells in the absence of anti-differentiation factors, ESCs spontaneously aggregate to form EBs. Such spontaneous formation is often accomplished in bulk suspension cultures whereby the dish is coated with non-adhesive materials, such as agar or hydrophilic polymers, to promote the preferential adhesion between single cells, rather than to the culture substrate. As hESC undergo apoptosis when cultured as single cells, EB formation often necessitates the use of inhibitors of the rho associated kinase (ROCK) pathway, including the small molecules Y-27632 and 2,4 disubstituted thiazole (Thiazovivin/Tzv). Alternatively, to avoid dissociation into single cells, EBs can be formed from hESCs by manual separation of adherent colonies (or regions of colonies) and subsequently cultured in suspension. Formation of EBs in suspension