The cell cycle in pluripotent stem cells is notable for the brevity of the G1 phase, permitting rapid proliferation and reducing the duration of differentiation signal sensitivity associated with the G1 phase. Changes in the length of G1 phase are understood to accompany the differentiation of human embryonic stem cells (hESCs), but the timing and extent of such changes are poorly defined. Understanding the early steps governing the differentiation of hESCs will facilitate better control over differentiation for regenerative medicine and drug discovery applications. Here we report the first use of real-time cell cycle reporters in hESCs. We coexpressed the chromatin-decorating H2B-GFP fusion protein and the fluorescence ubiquitination cell cycle indicator (FUCCI)-G1 fusion protein, a G1 phase-specific reporter, in hESCs to measure the cell cycle status in live cells. We found that FUCCI-G1 expression is weakly detected in undifferentiated hESCs, but rapidly increases upon differentiation. hESCs in the G1 phase display a reduction in undifferentiated colony-initiating cell function, underscoring the relationship between G1 phase residence and differentiation. Importantly, we demonstrate inter- and intracolony variation in response to chemicals that induce differentiation, implying extensive cell-cell variation in the threshold necessary to alter the G1 phase length. Finally, gain of differentiation markers appears to be coincident with G1 phase lengthening, with distinct G1 phase profiles associated with different markers of early hESC differentiation. Our data demonstrate the tight coupling of cell cycle changes to hESC differentiation, and highlight the cell cycle reporter system and assays we have implemented as a novel avenue for investigating pluripotency and differentiation.