Changes in the transcriptome are a well-known driver of stem cell differentiation. However, the nuances of such changes, including alternative isoform usage, the production of regulatory non-coding RNAs (ncRNAs), and differences in transcription initiation events, lack thorough investigation. As all of these features are known to be crucial during the differentiation process, we here utilize novel technologies based on the 5’ capture (cap-trapper) technique, to assay them on a genome-wide scale. To this end the Wells lab, in collaboration with FANTOM6, has collected a large dataset on an induced pluripotent stem cell (iPSC) to myeloid progenitor to macrophage differentiation series. Making use of information on transcription initiation via CAGE (capped analysis of gene expression), and CFC-seq (cap-trap full length cDNA sequencing), a total of over 200 previously unannotated genes and 20,000 novel mRNA isoforms, were discovered. Most of these uncharacterized genes were expressed in iPSCs and lost during differentiation. Further analyses of putative enhancer and promoter regions identified from transcription initiation events, showed a shift in transcription factor binding motif diversity from enhancers to promoters throughout differentiation, indicating a high degree of enhancer activation plasticity in stem cells. As it has been established that active enhancers produce ncRNAs that can mediate their regulatory role, future efforts of the projects are directed towards disentangling the role of enhancer transcription in the maintenance of pluripotency. To this end we will utilize data on the mode of transcription as well as chromatin state to identify enhancers which are common to the exit from pluripotency. Our future work will characterize the ncRNAs produced from these regions with regards to transcript structure, RNA-DNA interactions and enhancer-promoter looping correlates. We hope to ultimately investigate the transcriptomic impact of enhancer RNA knockdown as well as characterize their mechanism of action in pluripotency.