During embryogenesis, cells differentiate and organise into spatially defined regions in response to varying patterns of signalling. The mechanisms of this self-organisation and the relevance of signal dynamics to patterning remain elusive, due to limited methods to control signalling spatiotemporally in embryogenesis models. Here, using a previously developed optogenetic system (optoWnt), David Schaffer and colleagues investigate how cells organise into distinct domains in response to Wnt signalling in two-dimensional human embryonic stem cell (hESC) cultures. First, the authors show that optoWnt stimulation induces hESC differentiation along a mesendoderm lineage. Then, the authors mix wild-type and optoWnt hESCs in a co-culture and observe that Wnt signalling in spatially mixed cell subpopulations is sufficient to induce cell segregation and self-organisation into distinct epithelial and mesenchymal domains. This cell segregation is dependent on an epithelial-to-mesenchymal-like transition and the activation of TGFβ signalling. Furthermore, using transcriptomic analysis, the authors show that the Wnt-induced cells trigger BMP and TGFβ signalling in neighbouring wild-type cells, suggesting there is signalling feedback between cell types, and priming of wild-type cells for subsequent cell specification. Overall, this study establishes an hESC model system to investigate signalling feedback mechanisms in human embryogenesis.