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Nerve growth factor (NGF) has demonstrated great benefit in the treatment of neurotrophic corneal ulcers. There is evidence for multiple modes of action in promoting corneal healing, but only indirect evidence exists for NGF's effects on limbal stem cells (LSCs). Understanding the role of NGF in LSC biology will improve our understanding of paracrine regulation of the limbal niche and the design of stem cell‐based therapies for conditions such as LSC deficiency. In this article, we studied the regulation of NGF signaling components during LSC differentiation and the role of NGF in LSC proliferation and maintenance of the stem cell phenotype. LSC differentiation was induced by prolonged (40 day) culture which resulted in a significant increase in cell size, decrease in colony‐forming efficiency and expression of putative LSC markers. A protein microarray measuring expression of 248 signaling proteins indicated the low affinity NGF receptor p75 NTR  to be the most downregulated protein upon differentiation. Further confirmation by Western blotting and real‐time quantitative polymerase chain reaction indicated that NGF and p75 NTR  are expressed in early LSC cultures and downregulated upon differentiation. LSC cultures grown in the presence of anti‐NGF antibody showed decreased colony‐forming efficiency, DNA replication and expression of putative LSC markers  ABCG2  and  C/EBPδ . Supplementation of LSC culture medium with NGF extended the life span of LSC cultures in vitro and increased the expression of putative LSC markers Δ Np63α  and  ABCG2 . Taken together, our data indicate that NGF signaling is a key promoter of LSC proliferation, colony‐forming efficiency, and a maintainer of the LSC phenotype.  Stem Cells   2019;37:139–149

The Role of Nerve Growth Factor in Maintaining Proliferative Capacity, Colony‐Forming Efficiency, and the Limbal Stem Cell Phenotype