BRACHYURY T retained H3K4me3 binding, and increased levels of FTY720 Src-bcr-Abl inhibitor full-length transcript were detected, indicating a switch to active transcription. The cardiac transcription factor NKX-2.5 was associated with H3K4me3 but did not produce significant full-length transcript in either pluripotent or mesodermal populations, indicating it was transcriptionally paused in both stages. In contrast, NKX-2.5 showed high levels of 39 transcript in definitive cardiomyocytes at two weeks. The NEUROD1 locus did not produce significant amounts of fulllength transcript in any of the populations observed. The NEUROD1 locus was associated with H3K4me3 in embryonic stem cells and had a marked loss of H3K4me3 in mesoderm. This indicates that NEUROD1 transitions from being paused in embryonic stem cells to silent in mesodermal cells. This transcriptional silencing of NEUROD1 is consistent with lineage commitment away from ectodermal derivatives, like neurons, during the induction of mesoderm. Suppression of ectoderm is also supported by the absence of 39 transcript for the ectodermal transcription factor, SOX1, during our differentiation protocol. Having verified that genes with well-established biological functions behave as expected in our system, we next computationally sorted all detectable protein-coding loci in the human genome into one of the three transcriptional states based on their association with H3K4me3 and full-length transcript abundance. We then determined how many loci were changing between these three states during differentiation from embryonic stem cells to mesoderm. Interestingly, the overall distribution of the 12,867 analyzed proteincoding loci among active, paused and silent states did not change significantly during the transition from embryonic stem cells to mesoderm. There were many offsetting changes, however, with 1526 loci changing state. Of those loci that change transcriptional state, most are either “priming�?from silent to paused, or “archiving�?from paused to silent. We next sought to determine if genes changing expression transition directly from an active to a silent state, or if they instead pass through a paused intermediate. Strikingly, we found an overwhelming 98.0 to 98.9% of genes changing expression are transitioning into or out from a paused intermediate. These data indicate that the paused transcriptional state is a crucial control waypoint for developmentally regulated loci, and that initiation and elongation are distinctly regulated in hESC differentiation. To better understand the physiological significance of these transcriptional changes from embryonic stem cells to mesoderm, the Gene Ontology database was used to categorize proteincoding loci by annotated function. Compared to the set of all genes, loci involved in multicellular organismal development had a much higher fraction that were transcriptionally paused in embryonic stem cells.