Human embryonic stem cell (hESC) lines are known to be morphologically and phenotypically heterogeneous. The functional nature and relationship of cells residing within hESC cultures, however, has not been evaluated because isolation of single hESCs is limited to drug or manual selection. Here we provide a quantitative method using flow cytometry to isolate and clonally expand hESCs based on undifferentiated markers, alone or in combination with a fluorescent reporter. This method allowed for isolation of stage-specific embryonic antigen-3–positive (SSEA-3⁺) and SSEA-3⁻ cells from hESC cultures. Although both SSEA-3⁺ and SSEA-3⁻ cells could initiate pluripotent hESC cultures, we show that they possess distinct cell-cycle properties, clonogenic capacity and expression of ESC transcription factors. Our study provides formal evidence for heterogeneity among self-renewing pluripotent hESCs, illustrating that this isolation technique will be instrumental in further dissecting the biology of hESC lines.