Human ES cells (hESCs), due to their pluripotent nature, represent a particularly relevant model system to study the relationship between the replication program and differentiation state. In these studies AECC member Schildkraut defined the basic properties of the replication program in hESCs and compared them to the programs of hESC-derived multipotent cells (neural rosette cells) and primary differentiated cells (microvascular endothelial cells (MECs). Three genomic loci were characterized: two pluripotency regulatory genes POU5F1 (OCT4) and NANOG, and the IGH locus that is transcriptionally active specifically in B linage cells. Applying a high-resolution approach to capture images of individual replicated DNA molecules he demonstrated that for the loci studied, several basic properties of replication, including the average speed of replication forks and the average density of initiation sites, were conserved among the cells analyzed. He also demonstrated, for the first time, the presence of initiation zones in hESCs. However, significant differences were evident in other aspects of replication for the DNA segment containing the POU5F1 gene: The location of centers of initiation zones, and the direction of replication fork progression through the POU5F1 gene, were conserved in two independent hESC lines but were different in hESC-derived multipotent cells and MECs. Hence, these data identified features of the replication program characteristic of hESCs and defined specific changes in replication during hESC differentiation.