Pluripotent stem cells (PSC) are a tantalizing prospect for a renewable source of patient-specific hematopoietic stem cells (HSC), however efforts to obtain PSC derived HSC capable of long-term engraftment have largely failed. We set out with the primary aim of identifying novel molecular signatures of definitive hematopoiesis, so that these signatures could be applied to improve generation and isolation of HSC <italic>in vitro</italic>. Toward this end we pursued both discovery and application based strategies centered on <italic>Runx1</italic>; a transcription factor that is critical for the development of definitive HSC. The discovery arm identified epigenetic modifications at <italic>Runx1</italic> cis-regulatory elements that temporally associate with the transition from primitive to definitive hematopoiesis <italic>in vivo</italic>. We replicated these signatures <italic>in vitro</italic> by overexpressing HOXB4 in hematopoietic progenitors derived from murine embryonic stem cells (ESC), and found that HOXB4 directly interacts with the definitive-specific distal <italic>Runx1</italic> promoter and mediates increased transcription, loss of DNA methylation, and acquisition of active histone modifications at this locus. </DISS_para> <DISS_para>We next applied our understanding of <italic>Runx1</italic> regulation to generate a panel of clonal mESC lines harboring targeted, single-copy fluorescent reporters under the transcriptional control of <italic>Runx1</italic> cis-regulatory elements. These lines were used to interrogate the hematopoietic activity of each element independent of copy number and chromosomal position, allowing us to identify combinations that provided optimal activity and fidelity. Building upon this, we established mESC lines harboring synthetic fluorescent and bioluminescent mini genes replicating the structure of the endogenous <italic>Runx1</italic> locus and demonstrated that these lines reflect the dynamic promoter switching that occurs at <italic>Runx1</italic> during hematogenesis. Sub-fractionation of embryoid body cells based on promoter activity revealed that nearly all colony forming cells (CFC) reside in the distal promoter expressing fraction. With this information we identified specific conditions that could further mature and expand distal positive cells. Collectively, this work identified a previously undescribed molecular signature of definitive hematopoiesis and the mechanism by which it is established. In addition, we applied this knowledge to generate tools with which to interrogate hematopoietic development <italic>in vitro</italic>, and have demonstrated their utility in optimizing strategies for obtaining definitive hematopoietic progenitors from PSC.