is amenable to the formation of large quantities of EBs, but provides little control over the size of the resulting aggregates, often leading to large, irregularly shaped EBs. As an alternative, the hydrodynamic forces imparted in mixed culture platforms increase the homogeneity of EB sizes when ESCs are inoculated within bulk suspensions. Formation of EBs can also be more precisely controlled by the inoculation of known cell densities within single drops (10-20 µL) suspended from the lid of a Petri dish, known as hanging drops. While this method enables control of EB size by altering the number of cells per drop, the formation of hanging drops is labor-intensive and not easily amenable to scalable cultures. Additionally, the media can not be easily exchanged within the traditional hanging drop format, necessitating the transfer of hanging drops into bulk suspension cultures after 2–3 days of formation, whereby individual EBs tend to agglomerate. Recently, new technologies have been developed to enable media exchange within a modified hanging drop format. In addition, technologies have also been developed to physically separate cells by forced aggregation of ESCs within individual wells or confined on adhesive substrates, which enables increased throughput, controlled formation of EBs. Ultimately, the methods used for EB formation may impact the heterogeneity of EB populations, in terms of aggregation kinetics, EB size and yield, as well as differentiation trajectories. Differentiation within EBs Within the context of ESC differentiation protocols, EB formation is often used as a method for initiating spontaneous differentiation toward the three germ lineages. EB differentiation begins with the specification of the exterior cells toward the primitive endoderm phenotype. The cells at the exterior then deposit extracellular matrix (ECM), containing collagen IV and laminin, similar to the composition and structure of basement membrane. In response to the ECM deposition, EBs often form a cystic cavity, whereby the cells in contact with the basement membrane remain viable and those at the interior undergo apoptosis, resulting in a fluid-filled cavity surrounded by cells. Subsequent differentiation proceeds to form derivatives of the three germ lineages. In the absence of supplements, the “default” differentiation of ESCs is largely toward ectoderm, and subsequent neural lineages. However, alternative media compositions, including the use of fetal bovine serum as well as defined growth factor additives, have been developed to promote the differentiation toward mesoderm and endoderm lineages. As a result of the three-dimensional EB structure, complex morphogenesis occurs