Differences in cell responsiveness to an inductive signal contribute to the emergence of a variety of tissue types during animal development. In ascidian embryos, the Fibroblast Growth Factor (FGF) signal secreted from endoderm cells induces several different tissue types, such as notochord, mesenchyme and brain, at different positions in the embryo at the 32-cell stage. We show here in Halocynthia roretzi that FoxA and Zic are required for notochord formation in cells that receive the FGF signal. We also show that these transcription factors, only when both are supplied, are able to induce ectopic expression of the brachyury gene, a notochord-specific marker, in cells of all the three germ layers in an FGF-dependent manner. These results suggest that FoxA and Zic confer notochord-specific responsiveness to FGF signaling. Further analyses including knockdown and over-expression experiments showed that combinatorial inputs from maternally supplied and zigotically activated factors lead to overlapping expression of FoxA and Zic in the presumptive notochord cells, which eventually activate the expression of the brachyury gene in cooperation with FGF signaling. Our data illustrate how a complex gene network specifies the notochord at its specific position within the embryo.