The limbal niche supports the self-renewal of limbal epithelial stem cells (LESCs). The corneal stromal stem cell (CSSC) is an important component in the niche that regulates the LESC phenotype. However, the intercellular communication between LESCs and CSSCs has yet to be elucidated.A traditional two-dimensional (2D) system, a direct three-dimensional (3D) system, and an indirect 3D coculture system of LESCs and CSSCs were used to elucidate the paracrine pathway effect of CSSCs on LESCs. To reveal the impact of CSSC derived extracellular vesicles (CSSC-EVs) on LESCs, GW4869 and CSSC-EVs were added separately to the LESC culture medium. The outgrowth rate, cell density, differentiation, and stemness maintenance were compared among these methods. The miRNAs in the CSSC-EVs were sequenced, and the targeted Notch pathway was further confirmed by RT‒qPCR and Western blotting.Compared with 2D culture, both the direct and indirect 3D coculture systems yielded a higher outgrowth rate and expression of stem cell markers of LESCs. The phenotypes of LESCs cultivated using the two coculture approaches were also comparable. Nevertheless, GW4869 inhibited the effect of CSSCs on LESCs, and the addition of CSSC-EVs to the 2D culture system could increase cell density, and the proportion of p63αbright cells, which indicated that CSSC-EVs were crucial in regulating LESCs. Furthermore, the EV-AlixKD with reduced miRNA partly lost its regulating function. The abundant miRNAs in CSSC-EVs, such as hsa-miR-663b, hsa-miR-16-5p, and hsa-miR-1290, target the Notch pathway. The LESCs transfected with miR-663b had higher p63 expression via downregulating of the Notch pathway.CSSC-EV played an important role in promoting LESC proliferation and stemness maintenance by targeting Notch signaling via miRNAs, which will increase our understanding of the limbal niche and provide a potential new approach for LESC culture and the treatment of corneal epithelial disorders.