Limbal stem cells (LSCs) derived from human embryonic stem cells (hESCs) hold great potential for cell-based therapies for ocular surface diseases. The cell-based therapies mainly depend on an appropriate differentiation proposal with a high efficiency and on a suitable scaffold. In this study, we aimed to establish a feasible and efficient strategy for inducing hESCs into LSC-like cells by the LSC conditioned medium. The induced cells possessed the similar morphologic characteristics and expression of normal LSCs and showed a strong clonogenic and proliferative capacity in vitro. To construct a tissue-engineering corneal graft, these differentiated cells were seeded on an acelluar porcine corneal matrix (APCM), which maintained the corneal basement membrane in vitro. After 14 days culture, these induced cells gave rise to stratified epithelial cell sheets on the APCM and the basal cells still kept LSC characteristics. In rabbit total limbal stem cell deficiency (LSCD) models, the tissue-engineering graft had the potential to reconstruct the damaged ocular surface and alleviated the invasion of corneal neovascularization. These findings indicated that the engraftment constructed with the APCM scaffold and hESC-derived LSCs might be a potential therapy option for ocular surface regeneration in LSCD cases.