Development of many organs, including the mammary gland, involves ductal morphogenesis. Mammary ducts are bi-layered tubular structures comprising an outer layer of cap/myoepithelial cells (MECs) and an inner layer of luminal epithelial cells (LECs). Slit2 is expressed by cells in both layers,with secreted SLIT2 broadly distributed throughout the epithelial compartment. By contrast, Robo1 is expressed specifically by cap/MECs. Loss-of-function mutations in Slit2 and Robo1 yield similar phenotypes, characterized by disorganized end buds (EBs) reminiscent of those present in Ntn1-/- glands, suggesting that SLIT2 and NTN1 function in concert during mammary development. Analysis of Slit2-/-;Ntn1-/- glands demonstrates an enhanced phenotype that extends through the ducts and is characterized by separated cell layers and occluded lumens. Aggregation assays show that Slit2-/-;Ntn1-/- cells, in contrast to wild-type cells, do not form bi-layered organoids, a defect rescued by addition of SLIT2. NTN1 has no effect alone, but synergistically enhances this rescue. Thus, our data establish a novel role for SLIT2 as an adhesive cue,acting in parallel with NTN1 to generate cell boundaries along ducts during bi-layered tube formation.