Developmental fates are determined in relation to other fates in surrounding cells within the embryo, creating diversified tissue or cell types in the course of development. In ascidian embryos, maternally localized factors and inductive signals play essential and cooperative roles in fate determination at appropriate spatial and temporal positions during embryogenesis. Here, to clarify fate determination mechanisms of mesenchyme and muscle during mesenchyme induction by the fibroblast growth factor (FGF) signal in Halocynthia roretzi, we examined the function of transcription factor Tbx6, which acts as a pivotal mediator of the maternally localized muscle differentiation determinant Macho-1. Our results suggest that the level of Tbx6 expression increases in muscle lineage cells through positive feedback regulation that promotes muscle differentiation as well as mesenchymal fate suppression. In addition, the FGF signal inactivated Tbx6 transcriptional activity and positive feedback, leading to induction of the mesenchymal lineage. Taken together, our finding suggests that Tbx6 is an important factor for determining mesenchyme and muscle fates.