Follicular thyroid cells in vertebrates are considered to have evolved from the endostyle of invertebrate chordates based on the shared features of the concentration of iodine and expression of thyroid-related genes, including Nkx2-1, FoxE, and TPO. We previously demonstrated using gene knockout (KO) experiments on the ascidian Ciona robusta that the thyroid-related transcription factors, Nkx2-1 and FoxE, were associated with endostyle development, and also that Nkx2-1 regulated the expression of TPO via FoxE, even though the expression of Pax2/5/8 homologs was not affected. In the present study, which involved detailed observations of Ciona Nkx2-1-KO and FoxE-KO specimens, we found that ectopic bulged structures neighboring the hypoplastic endostyle occasionally emerged. In situ hybridization revealed that the ectopic bulged structures expressed TPO and Pax2/5/8 homologs. Further KO experiments on Ciona Pax2/5/8 homologs revealed no effects on endostyle development or TPO expression. These results suggest that, in addition to the established "Nkx2-1 -> FoxE-FoxE -> TPO" pathway, the ventral pharynx offers an insight into a potential relationship between Pax2/5/8 homologs and TPO. Accordingly, ascidians may show that the ventral pharyngeal endoderm possessed multiple gene regulatory networks for TPO expression prior to the evolutionary divergence leading to the vertebrate follicular thyroid.