AbstractBackground: Understanding the process by which pancreatic beta‐cells acquire their “fate” is critical to the development of in vitro directed differentiation protocols for cell replacement therapies for diabetics. To date, these efforts are hampered by a paucity of markers that distinguish pancreatic endocrine cells at different stages of differentiation. Results: Here, we identify EphB3 as a novel pro‐endocrine marker and use its expression to track delaminating islet lineages. First, we provide a detailed developmental expression profile for EphB3 and other EphB family members in the embryonic pancreas. We demonstrate that EphB3 transiently marks endocrine cells as they delaminate from the pancreatic epithelium, prior to their differentiation. Using a Tet‐inducible EphB3rtTA‐lacZ reporter line, we show that short‐term pulse‐labeled EphB3+ cells co‐express Pdx1, Nkx6.1, Ngn3, and Synaptophysin, but not insulin, glucagon, or other endocrine hormones. Prolonged labeling tracks EphB3+ cells from their exit from the epithelium to their differentiation. Conclusions: These studies demonstrate that pro‐endocrine cell differentiation during late gestation, from delamination to maturation, takes approximately 2 days. Together, these data introduce EphB3 as a new biomarker to identify beta‐cells at a critical step during their step‐wise differentiation and define the timeframe of endocrine differentiation. Developmental Dynamics 241:1008–1019, 2012. © 2012 Wiley Periodicals, Inc.