The kupffer’s vesicle (KV) is the so-called left-right organizer in teleost fishes. KV is formed from dorsal forerunner cells (DFCs) and generates asymmetrical signals for breaking symmetry of embryos. It is unclear how DFCs or KV cells are prevented from intermingling with adjacent cells. In this study, we show that the Eph receptor gene ephb4b is highly expressed in DFCs while the ephrin ligand genes including efnb2b are expressed in cells next to the DFCs cluster during zebrafish gastrulation. ephb4b knockdown or mutation and efnb2b knockdown cause dispersal of DFCs, a smaller KV and randomization of laterality organs. The DFCs often dynamically form blebs, filopodia and lamellipodia at the interface, which attempt to invade but are bounced back by adjacent non-DFC cells during gastrulation. Upon inhibition of Eph-ephrin signaling, however, the repulsion between DFCs and non-DFC cells is lost, allowing DFCs to migrate away. Ephb4b/efnb2b signaling by activating RhoA activity mediates contact and repulsion between DFCs and neighboring cells during gastrulation, preventing intermingling of different cell populations. Therefore, our data uncover an important role of Eph/ephrin signaling in maintaining DFCs boundary and KV boundary for normal left-right asymmetrical development.