To investigate the differential expression of stem cell factor (SCF) and thymic stromal lymphopoietin (TSLP) and their correlation to mast cells, between patients diagnosed with superior limbic keratoconjunctivitis (SLK) and normal subjects. A laboratory investigation included 22 surgical specimens of the superior bulbar conjunctiva from 17 patients with medically refractory SLK and 5 control subjects who underwent cataract or retinal surgery. Protein expression of tryptase, SCF, and TSLP in conjunctival specimens was detected by immunohistochemistry. The number of mast cells was correlated with immunohistochemistry intensity of SCF and TSLP. In patients with SLK, higher immunostaining intensity of SCF and TSLP was found in the conjunctival epithelium than that in the conjunctival subepithelial stroma. SCF and TSLP staining in the conjunctival epithelium was significantly more intense in patients with SLK than in normal subjects. In addition, there was a significant correlation between the number of tryptase (+) mast cells in conjunctival subepithelial stroma and TSLP immunointensity in the conjunctival epithelium and subepithelial stroma. Overexpression of SCF and TSLP was found in the conjunctival epithelium of patients with SLK. Significant correlation between TSLP grading and the number of mast cells was also found. SCF and TSLP may be involved in promoting mast cell migration and activation contributing to the pathogenesis of SLK.