Purpose: To evaluate in vitro the effect of nerve growth factor (NGF) on the differentiation of mouse corneal limbal progenitor cells into goblet cells and to observe the expression of mucin-5AC (MUC5AC) mRNA. Methods: Mouse limbal epithelial cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12 in vitro, and 63-kDa protein (p63) in cultured cells was identified with immunofluorescence staining. Different groups of the cultured cells were exposed to NGF at different concentrations (0 ng/ml, 10 ng/ml, 100 ng/ml, and 250 ng/ml). MUC5AC gene expression (real-time PCR) and goblet cell differentiation (MUC5AC immunofluorescence staining) were analyzed at different time points (24 h, 72 h, and 5 d). Results: In primary culture, the limbal epithelial cells were compact, uniform, and cobblestone pavement in shape. Some limbal epithelial cells were positive for p63. The MUC5AC-positive cells were detected when the cells were treated with 100 ng/ml NGF at each time point and with 250 ng/ml NGF at 5 d. The expression of MUC5AC mRNA increased when using 100 ng/ml NGF. The MUC5AC-positive cells were not detected when 0 ng/ml and 10 ng/ml NGF were used at each time point. Conclusions: The results of this study suggest that NGF might promote the differentiation of corneal limbal progenitor cells into conjunctival goblet cells and upregulate the expression of MUC5AC mRNA in primary culture. Further studies using an animal model in vivo are needed.