The temporal order in which segments of the genome are duplicated is referred to as the replication timing (RT) program. RT is established in each cell cycle coincident with the repositioning and anchorage of chromosomes in early G1. In general, segments that replicate in early S are organized into transcriptionally permissive chromatin, and segments that replicate in late S are assembled into repressive chromatin. During human embryonic stem cell (hESC) differentiation, segments of the genome undergo changes in RT, which are accompanied by changes in chromatin compartments, and transcriptional activity. Determining the order these changes occur during hESC differentiation required defining cell cycle parameters for hESCs. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) system is incapable of demarcating G1/S cell cycle transitions. Instead, we employed a combination of fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replicating DNA and track 3D organization. We find that re-localization and anchorage of chromosomes were completed prior to the onset of S phase, even in the context of an abbreviated G1 phase. Furthermore, we find that single hESCs preferentially differentiate from G1. We show changes in RT are remarkably coincident with transcription; although, neither is sufficient for the other to occur. We also show changes in RT accompany cell commitment during the first cell cycle and precede changes in chromatin compartments. Finally, we find that in hESCs, domains that switch from early to late replication interact more frequently with late replicating chromatin, suggesting hESCs may be poised to quickly repress early to late switching domains upon stimulation.