Although embryonic stem (ES) cells have the regenerative capability of producing any tissue in our body, the development of efficient methods to generate a large number of ES cells with high purity has still remained a technical challenge before they can be used routinely in clinical trials. In this chapter, we describe a simple procedure to establish an ES cell 3D culture system through alginate-poly-l -lysine-alginate (APA) microencapsulation. The APA microcapsule provides an environment that promotes better cell growth and differentiation without requiring feeder layers.