It has been clearly demonstrated that DNA methyltransferase inhibition can induce embryonic stem cells to differentiate into cells of specific tissues by altering the genes expression. Chicken embryonic fibroblasts (CEFs) are mesenchymal stem cell (MSC)-like multipotent progenitor cells and can both self-renew and undergo differentiation. In this study, we hypothesize that DNA demethylation may lead to cell differentiation and the re-expression of development-related genes in chicken embryo fibroblasts. Thus, we investigated which development-related genes are affected when CEFs are treated with DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine (5-Aza-dC) and how those genes are affected. In this study, we analyzed the expression profiles of genes by RNA sequencing of CEFs treated with 5-Aza-dC. A total of 40 differentially expressed genes (DEGs) (>2-fold change) related to chicken development were identified. Co-expression cluster analysis revealed that these genes are mainly related to development of bone, feather follicle, epidermis, and blood vessels. Moreover, the molecular interaction network analysis showed that canonical Wnt signaling tended to play a central role in development associated with these genes. These findings will help us to understand the molecular regulatory mechanisms of development.