An important step in early embryonic development is the allocation and segregation of germ layer fates into distinct embryonic regions. However, the mechanism that segregates the mesendoderm into mesoderm and endoderm fates remains largely unknown in most animals. Here, using ascidians, a primitive chordate, we show that these fates are segregated by partitioning of asymmetrically localized Not mRNA from the mesendoderm cell to its mesodermal daughter. Migration of the mesendoderm cell nucleus to the future mesoderm-forming region, release of Not mRNA from the nucleus, Wnt5α-dependent local retention of the mRNA, and subsequent repositioning of the mitotic spindle to the center of the cell are each required for the asymmetric localization and partitioning of Not mRNA. Our results show that nuclear migration plays an unexpected role in asymmetric cell divisions that segregate germ layer fates in chordate embryos.