AbstractThe neuroectoderm is patterned along a rostral-caudal axis in response to localized factors in the embryo, but exactly how these factors act as positional information for this patterning is not yet fully understood. Here, using the self-organizing properties of mouse embryonic stem cell (ESC), we report that ESC-derived neuroectoderm self-generates a Six3⁺ rostral and a Irx3⁺ caudal bipolarized patterning. In this instance, localized Fgf signaling performs dual roles, as it regulates Six3⁺ rostral polarization at an earlier stage and promotes Wnt signaling at a later stage. The Wnt signaling components are differentially expressed in the polarized tissues, leading to genome-wide Irx3⁺ caudal-polarization signals. Surprisingly, differentially expressed Wnt agonists and antagonists have essential roles in orchestrating the formation of a balanced rostral-caudal neuroectoderm pattern. Together, our findings provide key processes for dynamic self-patterning and evidence that a temporally and locally regulated interaction between Fgf and Wnt signaling controls self-patterning in ESC-derived neuroectoderm.