The cornea is continually regenerated to maintain clear vision, due to a population of stem cells located in the limbal region between the cornea and the conjunctiva. These limbal stem cells (LESC) reside in crypts in structures called the Pallisades of Vogt. This LESC niche has a specific stromal matrix and cells that are on close contact with LESC. Among these stromal cells are limbal epithelial cells that help form the niche and limbal melanocytes (LM) that have been suggested to protect LESC via antioxidant production. In this paper human limbal melanocytes were prepared from cadaveric biopsies and used in culture with limbal epithelial cells (LEC) from the same source. LEC grown on LM formed large colonies that contained cells with stem cell markers and characteristics. In 3D cultures LM coculture induce multilayered epithelial structures. These results strongly support a role for limbal melanocytes in the maintenance of the LESC niche through interaction with LEC.