Haematopoiesis is the process by which blood cells are continuously replenished during the course of an organism’s lifetime. Definitive haematopoiesis involves the production of haematopoietic stem and progenitor cells (HSPCs). HSPCs sustain blood production in all vertebrates. HSPCs are specified from the dorsal aorta during embryonic development, through a process known as endothelial-to-haematopoietic transition (EHT). Following HSPC specification from the aorta, HSPCs migrate to a subsequent niche, where they receive highly regulated signals from the microenvironment, allowing them to expand. Gaining a deeper understanding of the molecular cues required for HSPC specification and expansion in the vertebrate embryo, may allow translation of these principles into improved success of in vitro generation and ex vivo expansion of HSPCs. Therefore, this holds great promise for regenerative medicine, including gene therapy. In my thesis, I investigated molecular players involved in HSPC specification and expansion, predominantly using the zebrafish embryo as a model system.