Haematopoietic stem cells (HSCs) support maintenance of the haematopoietic and immune systems throughout the life of vertebrates, and are the therapeutic component of bone marrow transplants. Understanding native specification of HSCs, to uncover key signals that might help improve in vitro directed differentiation protocols, has been a long-standing biomedical goal. The current impossibility of specifying true HSCs in vitro suggests that key signals remain unknown. We speculated that such signals might be presented by surrounding 'niche' cells, but no such cells have been defined. Here we demonstrate in zebrafish, that trunk neural crest (NC) physically associate with HSC precursors in the dorsal aorta (DA) just prior to initiation of the definitive haematopoietic program. Preventing association of the NC with the DA leads to loss of HSCs. Our results define NC as key cellular components of the HSC specification niche that can be profiled to identify unknown HSC specification signals.