Human embryonic stem (ES) cells can undergo spontaneously differentiation in standard culture conditions, demonstrating that the undifferentiated state is relatively unstable. The heterogeneous expression of SSEA3 observed within human ES colonies, provides a means to examine undifferentiated stem cell substates. Through functional testing of single cells we have shown that undifferentiated ES cells can be segregated into functionally discrete subpopulations on the basis of SSEA3 expression: SSEA3(High), SSEA(Low) and SSEA3(Negative). Human ES subpopulations were found to be interconvertible, but they possess distinct properties when challenged to differentiate along the neural lineage. These data suggest that ES cells with pluripotent/self-renewal capacities can exhibit different responses to induction of differentiation.