during EB differentiation, including the appearance of both epithelial- and mesenchymal-like cell populations, as well as the appearance of markers associated with the epithelial-mesenchymal transition (EMT). Additionally, the inductive effects resulting from signaling between cell populations in EBs results in spatially and temporally defined changes, which promote complex morphogenesis. Tissue-like structures are often exhibited within EBs, including the appearance of blood islands reminiscent of early blood vessel structures in the developing embryo, as well as the patterning of neurite extensions (indicative of neuron organization) and spontaneous contractile activity (indicative of cardiomyocyte differentiation) when EBs are plated onto adhesive substrates such as gelatin. More recently, complex structures, including optic cup-like structures were created in vitro resulting from EB differentiation. Parallels with embryonic development Much of the research central to embryonic stem cell differentiation and morphogenesis is derived from studies in developmental biology and mammalian embryogenesis. For example, immediately after the blastocyst stage of development (from which ESCs are derived), the embryo undergoes differentiation, whereby cell specification of the inner cell mass results in the formation of the hypoblast and epiblast. Later on in postimplantation development, the anterior-posterior axis is formed and the embryo develops a transient structure known as the primitive streak. Much of the spatial patterning that occurs during the formation and migration of the primitive streak results from the secretion of agonists and antagonists by various cell populations, including the growth factors from the Wnt and transforming growth factor β (TGFβ) families (Lefty 1, Nodal), as well as repressors of the same molecules (Dkk-1, Sfrp1, Sfrp5). Due to the similarities between embryogenesis and ESC differentiation, many of the same growth factors are central to directed differentiation approaches. In addition, advancements of EB culture resulted in the development of embryonic organoids (Gastruloids) which show remarkable parallels to embryonic development such as symmetry-breaking, localised brachyury expression, the formation of the embryonic axes (anteroposterior, dorsoventral and Left-Right) and gastrulation-like movements. Challenges to directing differentiation In contrast to the differentiation of ESCs in monolayer cultures, whereby the addition of soluble morphogens and the extracellular microenvironment can be precisely and homogeneously controlled, the three-dimensional structure of EBs poses challenges to directed differentiation. For example, the visceral endoderm population which forms the exterior of EBs, creates an exterior “shell” consisting of tightly connected epithelial-like cells, as well as a dense ECM. Due to such physical restrictions, in combination with EB size, transport limitations occur within