The intestinal epithelium is one of the most rapidly self-renewing tissues throughout life in mammals. A small population of stem cells at the base of crypt in the epithelium can continually self-renew and give rise to differentiated epithelial cells. The self-renewal and differentiation of intestinal stem cells are under a tight control during homeostasis, and disruption of this balancing regulation leads to intestinal degeneration or tumorigenesis. Accordingly, exploration of the mechanism underlying the regulation of stem cells is essential for the understanding and treatment of intestinal disorders. As traditional methods using mice models are costly and time-consuming, the recently established ex vivo intestinal organoids model provides an ideal tool to investigate the mechanisms regulating the self-renewal and differentiation of intestinal stem cells. The intestinal organoids recapitulate major characteristics in both structure and function of intestinal epithelium in vivo. Here, we describe a new protocol to generate the intestinal organoids from both crypts and single stem cells with a higher efficiency using the small molecule blebbistatin and provide an approach to assess the self-renewal and differentiation of stem cells in intestinal organoids.