Ascidian Ciona intestinalis tadpole larvae exhibit left-right asymmetry. The photoreceptors are situated on the right side of the sensory vesicle, and the tail curls along the left side of the trunk within the chorion. In tailbud embryos, the Ci-pitx gene is expressed in the left-side epidermis. It was previously reported that embryos generated from naked eggs, which lack the chorionic membrane and accessory cells (follicle cells attached to the outside of the chorion and test cells covering the inner surface of the chorion), show bilateral expression of Ci-pitx. This suggested that the chorion or accessory cells are needed for generation of asymmetry. Here, we show that a brief treatment with 60% artificial seawater (ASW) before, but not after, the neurula stage results in bilateral expression of Ci-pitx in the chorion of tailbud embryos, loss of follicle cells, and randomization of both the direction of tail curling and the locations of photoreceptors in larvae. This treatment also impaired the transient counterclockwise rotation within the chorion at the neurula stage. Nearly all test cells in the chorion died following 60% ASW treatment. These results suggest that dead test cells blocked the neural rotation and impaired left-right asymmetry. We also showed that tailbud embryos and larvae generated from defolliculated eggs produced by 80% ASW treatment, in which the test cells were alive, showed normal left-right asymmetry, suggesting that the follicle cells were not essential for asymmetric morphogenesis.