Mouse R-spondin3 (Rspo3) is a member of the R-spondin protein family, which is characterized by furin-like cysteine-rich domains and a thrombospondin type 1 repeat. Rspo3 has been proposed to function as a secretory molecule that promotes the Wnt/beta-catenin signaling pathway. We generated mice bearing a mutant Rspo3 allele in which a lacZ-coding region replaced the coding region of the first exon. The homozygous mutant mice died at about embryonic day 10, due to impaired formation of the labyrinthine layer of the placenta. Rspo3 was expressed in the allantoic component of the labyrinth. In the homozygous mutant placentas, fetal blood vessels did not penetrate into the chorion, and expression of Gcm1, encoding the transcription factor glial cells missing-1 (Gcm1), was dramatically reduced in the chorionic trophoblast cells. These findings suggest a critical role for Rspo3 in the interaction between chorion and allantois in labyrinthine development.