Despite deep evolutionary roots in the metazoa, the gene regulatory network driving germ layer specification is surprisingly labile both between and within phyla. In Xenopus laevis, SoxB1- and SoxF-type transcription factors are intimately involved in germ-layer specification, in part through their regulation of Nodal signaling. However, it is unclear if X. laevis is representative of the ancestral vertebrate condition, as the precise roles of SoxF and SoxB1 in germ-layer specification vary among vertebrates, and there is no evidence that SoxF mediates germ-layer specification in any invertebrate. To better understand the evolution of germ-layer specification in the vertebrate lineage, we analyzed the expression of soxB1 and soxF genes in embryos and larvae of the basal vertebrate lamprey, and the basal chordate amphioxus. We find that both species maternally deposit soxB1 mRNA in the animal pole, soxF mRNA in the vegetal hemisphere, and zygotically express soxB1 and soxF throughout nascent ectoderm and mesendoderm, respectively. We also find that soxF is excluded from the vegetalmost blastomeres in lamprey and that, in contrast to vertebrates, amphioxus does not express soxF in the oral epithelium. In the context of recent work, our results suggest that a maternally established animal/vegetal Sox axis is a deeply conserved feature of chordate development that predates the role of Nodal in vertebrate germ-layer specification. Furthermore, exclusion of this axis from the vegetal pole in lamprey is consistent with the presence of an extraembryonic yolk mass, as has been previously proposed. Finally, conserved expression of SoxF in the forming mouth across the vertebrates, but not in amphioxus, lends support to the idea that the larval amphioxus mouth is nonhomologous to the vertebrate mouth.