Individual stem cells are functionally defined by their self-renewal and differentiation potential. Methods for clonal analysis are essential for understanding stem cells, particularly given the increasing evidence for stem-cell heterogeneity. Stem cells reside within complex microenvironments, making single-cell analysis particularly challenging. Furthermore, simultaneous molecular and functional characterization of single stem cells is not trivial. Here we explore clonal assays applied to stem cell biology and their use in understanding the cellular and molecular basis of stem-cell identity.