Definitive hematopoietic stem cells develop from the ventral wall of the dorsal aorta (hemogenic endothelium) in response to shear stress (blood flow) via a de-differentiation process known as endothelial-to-hematopoietic transition (EHT). Only ~50 HSCs are generated per embyro but these are sufficient to sustain the hematopoieitic system for life. We have employed transgenic zebrafish which express two reporters, Tg(Kdrl:mCherry) and Tg(Myb:YFP), to trace 4 steps of EHT (endothelium, hemogenic endothelium, HSC and progenitor cell) by FACS and confocal microscopy. We undertook dynamic expression profiling (RNA-seq) of limiting numbers of FACS sorted cells (2000) using NexTera chemistry and sequencing on a NextSeq500 (Illumina). We sequenced 3 biological replicates of four time points (~700 million reads total) and mapped the sequence tags to the zebrafish genome (danRer7). We used Tophat and weighted gene correlation network analysis (WGCNA) in R to find clusters of co-regulated genes. We found remarkably robust but transient up-regulation of many transcription factors with known key roles in early HSC generation as determined by previous studies in mice and zebrafish models. These include the key transcription factors runx3, gata2b, bloody fingers, draculin, c-myb, sox17 and ikzf1 (Ikaros). Gata4, gata2a, gata2b and gata1 are dynamically expressed in the four populations, which suggests an extended ‘gata switch’ program drives EHT as it does erythropoiesis in the mouse. We found similar dynamic Klf and Sox gene networks. We found a very large number of genes involved in ribosome biogenesis are markedly up-regulated during EHT. Some of these have been recently implicated in generation of HSCs in mouse models but other genes are novel. We are in the process of validating these genes by WISH and eventually by CRISPR/Cas9 loss of function experiments. We will discuss the consequences of chemical inhibition of Pol1 on the EHT process.