Understanding of visual system function and the development of new therapies for corneal diseases and damages depend upon comprehension of the biological roles of the tissue. The in vitro cultivation of corneal epithelial cells and cell lines derived from them has become a powerful tool to analyze and understand such issues. Currently, researchers have developed well-defined and precisely described culture protocols and a collection of corneal epithelial cell lines. These cell lines have been obtained through different experimental approaches: (1) the ectopic expression of oncogenes, (2) the inactivation of p16 and p53 pathways and hTERT expression, and (3) the spontaneous establishment after serial cultivation of cells. The advantages or disadvantages for these approaches are discussed. In conclusion, the availability of several culture protocols and immortalized cell lines that express corneal epithelial phenotype will be useful for investigating issues such as gene regulation and tissue development, or for validating alternative methods in toxicology.