Abstract: The incessant requirement to replenish differentiated cells in tissues such as the gut and blood requires efficient signaling and transcriptional mechanisms to regulate the genesis and function of stem cells. It is of particular importance to elucidate mechanisms controlling master regulatory transcription factors that orchestrate stem cell levels/activities. We described an intronic cis-regulatory element (+9.5) required for expression of the master regulator of hematopoiesis GATA-2, establishment of the hematopoietic stem/progenitor cell compartment in the fetal liver, vascular integrity, and embryogenesis (Johnson et al. J Clin. Invest., 2012). Herein, we utilize a novel allele-specific assay to dissect molecular mechanisms underlying +9.5 element function. Targeted deletion of the endogenous +9.5 element selectively erased the active chromatin structure surrounding the element and strongly reduced Gata2 expression from the mutant allele in +9.5+/- fetal liver cells. A GATA switch that instigates Gata2 repression, involving GATA-1-mediated displacement of GATA-2 (Grass et al. PNAS, 2003), abrogated the active chromatin structure of the +9.5 element and two GATA switch sites not essential for Gata2 transcriptional activity in vivo. However, only the +9.5 element active chromatin was rapidly restored upon GATA-1 dissociation. Thus, the +9.5 element has a GATA factor-regulated tripartite chromatin signature (GATA-2-bound, active; GATA-1-bound, inactive; GATA-1-dissociated, active) reflecting its capacity to mediate dynamic chromatin structural transitions. This tripartite signature is predicted to be an essential molecular attribute endowing the +9.5 element, and analogous cis-regulatory elements, with uniquely important stem cell-regulatory activity. Studies are underway to discover the molecular determinants of the tripartite signature and to identify +9.5-like sites in the genome that share the crucial stem cell-regulatory activity and therefore constitute an “HSC Cistrome”.