How blood cells and ultimately hematopoietic stem cells (HSCs) arise from uncommitted cells during development is only vaguely understood. Especially the initial formation of the first blood precursors in lateral mesoderm has been difficult to dissect. Investigating these early mechanisms of blood cell fate determination with genetic model systems provides a vital starting point for therapeutic drug discovery and the targeted manipulation of iPS cells to treat hematologic diseases. The goal of the proposed research project is to elucidate the mechanisms that drive uncommitted embryonic cells into a hematopoietic cell fate by using the vertebrate zebrafish (Danio rerio). To visualize, isolate, and fate-map the earliest stages of hematopoietic cell formation, the proposed work applies transgenic zebrafish reporters under control of the newly isolated draculin gene promoter/enhancer. draculin is the earliest marker for the emergence of hematopoietic cells from lateral mesoderm during embryo development. The transgenic labeling with draculin will be harnessed for progenitor culture assays to uncover the earliest hematopoietic cell fate potential of uncommitted lateral mesoderm. These experiments will be complemented with in vivo fate mapping using Tamoxifen-controlled Cre/lox methods. The proposed study will also apply novel zebrafish microinjection approaches to test and identify transcription factor combinations that enforce blood cell fates and HSC formation. The preliminary results revealed a combination of transcription factors that ectopically induces a hematopoietic gene program in the embryo. Altogether, this work will break new ground in our understanding of the earliest blood-forming mechanisms, and provide new insight into how cells can be experimentally guided towards the therapeutically desirable HSC fate.