Efnb2 encodes for the ligand Ephrinb2 that binds to its cognate Eph receptor, with which plays an integral role in angiogenesis, stem cell regulation and tumorigenesis. Using a pituitary-specific Cre-driver (Hesx1Cre), we conditionally deleted Efnb2 from the early stem/progenitor cells (PSCs) of the developing pituitary gland. We found that Efnb2 is expressed in PSCs both during embryogenesis and adulthood, suggesting its involvement in PSCs maintenance. Here we show that genetic ablation of Efnb2 (Efnb2fl/fl;Hesx1Cre/+) results in severe pituitary hyperplasia, abnormal gland morphogenesis and delay in the terminal differentiation of hormone-producing cells. In order to understand the underlying molecular mechanisms by which Efnb2 deletion leads to pituitary abnormalities during embryogenesis, we performed transcriptomic analyses using mRNA-Seq at early stages of pituitary development on Efnb2+/+ and Efnb2−/− cell populations. Interestingly, unsupervised gene set enrichment analyses of the transcriptomic results identified Efnb2 as a key negative regulator of PSCs proliferation, whilst involved in the regulation of epithelial integrity and epithelial to mesenchymal transition (EMT). Functional assays performed to validate transcriptomic analyses revealed that Efnb2-null PSCs hyper-proliferate both in vitro and in vivo and downregulate the expression of genes involved in EMT, indicating a role of Efnb2 in these two developmental pathways. Indeed, whilst Efnb2-null PSCs within the marginal zone hyper-proliferate, they downregulate genes of epithelial integrity, resulting in an abnormal EMT transcriptomic profile. Additionally, these events leads to a significant delay of the pituitary cell lineage commitment program, determining severe reduction in the expression of Pit1, Pomc1 and Gsu commitment markers. Taken together, our results reveal a novel role for Efnb2 (Ephrinb2) as key regulator of pituitary gland development. Efnb2 negatively regulate the proliferation of pituitary stem/progenitor cells while controlling the epithelial integrity of the stem cell niche and regulating EMT.