In this chapter, we describe a simple and unique method for the differentiation of mouse embryonic stem cells into insulin-producing cells. In addition to cytokines and growth factors, key transcription factors for pancreatic development are applied in this method through protein transduction technology. Furthermore, a combination of nanofiber plates and laminin coatings improves the yield of differentiated cells. The insulin-producing cells derived through this method express marker genes of mature β-cells and have an ability to secrete insulin; therefore, these cells are useful for fundamental studies on pancreatic development, drug development, and regenerative medicine for diabetes.