Reprogramming of somatic cells to desired cell-types holds great promise in regenerative medicine. However, production of transplantable hematopoietic stem cells (HSCs) in vitro by defined factors has not yet been achieved. Therefore, it is critical to fully understand the molecular mechanisms of HSC development in vivo. Here we show that Fev, an ETS transcription factor, is a pivotal regulator of HSC development in vertebrates. In fev-deficient zebrafish embryos, the first definitive HSC population was compromised and fewer T cells were found in the thymus. Genetic and chemical analyses support a mechanism whereby Fev regulates HSC through direct regulation of ERK signaling. Blastula transplant assay demonstrates that Fev regulation of HSC development is cell autonomous. Experiments performed with purified cord blood show that fev is expressed and functions in primitive HSCs in humans, indicating its conserved role in higher vertebrates. Our data indicate that Fev-ERK signaling is essential for hemogenic endothelium-based HSC development.