In animal development, secreted signaling molecules evoke all-or-none threshold responses of target gene transcription to specify cell fates. In the chordate Ciona intestinalis, the neural markers Otx and Nodal are induced at early embryonic stages by Fgf9/16/20 signaling. Here we show that three additional signaling molecules act negatively to generate a sharp expression boundary for neural genes. EphrinA signaling antagonizes FGF signaling by inhibiting ERK phosphorylation more strongly in epidermal cells than in neural cells, which accentuates differences in the strength of ERK activation. However, even weakly activated ERK activates Otx and Nodal transcription occasionally, probably because of the inherently stochastic nature of signal transduction processes and binding of transcription factors to target sequences. This occasional and undesirable activation of neural genes by weak residual ERK activity is directly repressed by Smad transcription factors activated by Admp and Gdf1/3-like signaling, further sharpening the differential responses of cells to FGF signaling. Thus, these signaling pathways coordinate to evoke a threshold response that delineates a sharp expression boundary.