WNT4, a member of the Wnt family of ligands, is critical for the development of the female reproductive tract. Analysis of Wnt4 expression in the adult uterus during pregnancy indicates that it may play a role in the regulation of endometrial stromal cell proliferation, survival, and differentiation, which is required to support the developing embryo. To investigate the role of Wnt4 in adult uterine physiology, conditional ablation of Wnt4 using the PR cre mouse model was accomplished. Ablation of Wnt4 rendered female mice subfertile due to a defect in embryo implantation and subsequent defects in endometrial stromal cell survival, differentiation, and responsiveness to progesterone signaling. In addition to altered stromal cell function, the uteri of PR cre/+ Wnt4 f/f (Wnt4 d/d) mice displayed altered epithelial differentiation characterized by a reduction in the number of uterine glands and the emergence of a p63-positive basal cell layer beneath the columnar luminal epithelial cells. The altered epithelial cell phenotype was further escalated by chronic estrogen treatment, which caused squamous cell metaplasia of the uterine epithelium in the Wnt4 d/d mice. Thus, WNT4 is a critical regulator not only of proper postnatal uterine development, but also embryo implantation and decidualization.—Franco, HL, Dai, D., Lee, KY, Rubel, CS, Roop, D., Boerboom, D., Jeong, J.-W., Lydon, J.-P., Bagchi, IC, Bagchi, MK, DeMayo, FJ WNT4 is a key regulator of normal postnatal uterine development and progesterone signaling during embryo implantation and decidualization in the mouse.