Haematopoietic stem and progenitor cell (HSPC) transplant is a widely used treatment for life-threatening conditions including leukemia; however, the molecular mechanisms regulating HSPC engraftment of the recipient niche remain incompletely understood. Here, we developed a competitive HSPC transplant method in adult zebrafish, using in vivo imaging as a non-invasive readout. We used this system to conduct a chemical screen and identified epoxyeicosatrienoic acids (EET) as a family of lipids1,2 that enhance HSPC engraftment. EETs’ pro-haematopoietic effects were conserved in the developing zebrafish embryo, where 11,12-EET promoted HSPC specification by activating a unique AP-1/runx1 transcription program autonomous to the haemogenic endothelium. This effect required the activation of the PI3K pathway, specifically PI3Kγ. In adult HSPCs, 11,12-EET induced transcriptional programs, including AP-1 activation, which modulate multiple cellular processes, such as migration, to promote engraftment. Finally, we demonstrated that the EET effects on enhancing HSPC homing and engraftment are conserved in mammals. Our study established a novel method to explore the molecular mechanisms of HSPC engraftment, and discovered a previously unrecognized, evolutionarily conserved pathway regulating multiple haematopoietic generation and regeneration processes. EETs may have clinical application in marrow or cord blood transplantation.