ROBO1 is an axon guidance molecule that is expressed on pioneer axons in the neural tube and proliferative zones of the cortical plate. It plays an important role in axon pathfinding and tract development as well as in neuronal proliferation and dendrite branching. It is restricted to axonal growth cones that do not cross the midline and to midline-crossing axons only after they cross. ROBO1 binds to Slit1 and Slit2, leading to growth cone collapse and axonal branching. It also regulates Netrin-1 and Semaphorin 3A and 3F responsiveness through its ability to form in cis complexes with Neuropilin-1, Neuropilin-2, Plexin A1, DCC, and FLRT3. ROBO1-Slit2 interactions restrict branching morphogenesis in developing ductal epithelium, suppress tumorigenesis, enhance VEGF-induced angiogenesis, and enhance CXCL12 induced T cell chemotaxis by associating with CXCR4.