Human embryonic stem cells (hESCs) can be maintained in vitro as immortal pluripotent cells but remain responsive to many differentiation-inducing signals. Investigation of the initial critical events involved in differentiation induction would be greatly facilitated if a specific, robust, and quantitative assay for pluripotent hESCs with self-renewal potential were available. Here we describe the results of a series of experiments to determine whether the formation of adherent alkaline phosphatase-positive (AP(+)) colonies under conditions optimized for propagating undifferentiated hESCs would meet this need. The findings can be summarized as follows. (a) Most colonies obtained under these conditions consist of >or=30 AP(+) cells that coexpress OCT4, NANOG, SSEA3, SSEA4, TRA-1-60, and TRA-1-81. (b) Most such colonies are derived from SSEA3(+) cells. (c) Primary colonies contain cells that produce secondary colonies of the same composition, including cells that initiate multilineage differentiation in embryoid bodies (EBs). (d) Colony formation is independent of plating density or the colony-forming cell (CFC) content of the test population over a wide range of cell concentrations. (e) CFC frequencies decrease when differentiation is induced by exposure either to retinoic acid or to conditions that stimulate EB formation. Interestingly, this loss of AP(+) clonogenic potential also occurs more rapidly than the loss of SSEA3 or OCT4 expression. The CFC assay thus provides a simple, reliable, broadly applicable, and highly specific functional assay for quantifying undifferentiated hESCs with self-renewal potential. Its use under standardized assay conditions should enhance future elucidation of the mechanisms that regulate hESC propagation and their early differentiation.