The inability to identify, isolate, and culture intestinal epithelial stem cells (IESCs) has been prohibitive to the study and therapeutic utilization of these cells. Using a Sox9(EGFP) mouse model, we demonstrate that Sox9(EGFP) fluorescence signatures can be used to differentiate between and enrich for progenitors (Sox9(EGFPsubLo)) and multipotent IESCs (Sox9(EGFPlo)). Sox9(EGFPlo) cells generate "organoids" in a recently defined culture system that mimics the native IESC niche. These organoids possess all four differentiated cell types of the small intestine epithelium, demonstrating the multipotent capacity of Sox9(EGFPlo) cells. Our results are consistent with the previously reported observation that single IESCs generate cryptlike units without a detectable mesenchymal cell component. A prospective search revealed that CD24 is expressed in the Sox9(EGFPlo) population and marks IESCs that form organoids in culture. CD24 represents the first cell surface marker that facilitates fluorescence-activated cell sorting enrichment of IESCs with widely available antibodies without requiring a specialized fluorescent reporter gene mouse model.