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Endothelial to hematopoietic transition (EHT) is a crucial step in the formation of definitive hematopoietic stem cells during embryonic development. Holding such a critical developmental role not many details regarding molecular changes and cell surface marker (CSM) expression on these transitional cells are known. We think that sub-segregating the process of EHT holds great therapeutic potential by giving new indications to understand the hematopoietic stem cell maturation. We generated embryonic stem (ES) cell lines from Hoxb4-YFP reporter mice (Hills, 2011), in which YFP expression marks functional HSCs in adult and embryonic mice. When these cell lines were subject to embryoid body differentiation assays, we observed a transient Hoxb4/YFP+ cell population, which corresponded to the emergence of hemogenic endothelial cells in culture. We then analyzed the gene expression profile of the Hoxb4+ cells and compared it to immediate precursor (Hoxb4-Flk1+) and daughter (Hoxb4-CD41+) cell populations. As well as documenting the expression of numerous molecular markers previously associated with EHT, we also observed an elevated inflammatory gene expression signature that has previously been characterized as a mediator of hematopoietic specification in vivo. We were additionally able to identify 45 novel cell surface markers that could potentially be used to prospectively isolate and sub segregate cells undergoing EHT. 26 of these targets were subsequently verified using MRM mass spectrometry. To establish if any of these markers were functionally relevant, we generated knockout (KO) cell lines using CRISPR/Cas9. Upon EB differentiation, we observed a profound block in hematopoietic differentiation for ES cells that were KO for the membrane proteins Evi2a and Lyve1. This block was manifest at the EHT stage, as verified by time-lapse video imaging, and was equivalent in magnitude to Runx1 KO, suggesting a crucial role of Evi2a and Lyve1 during the EHT stage of hematopoietic development. Notably, this defect could be rescued by genetic replacement of the deleted gene. When comparing our data to human fetal liver data sets Evi2a is highly expressed in fetal liver, as well as in HSCs isolated from murine AGM and fetal liver.