Haematopoietic stem cells (HSCs) can differentiate into all blood cell types, making HSC transplantation a promising treatment for many blood-related disorders. Allogenic heathy HSCs can be transplanted into patients, although this approach is limited by availability and compatibility of the donor. Moreover, there are no protocols available to generate transplantable HSCs from pluripotent stem cells (PSC), or expand HSCs ex vivo, as the molecules involved in HSC ontogeny remain poorly understood. Embryonically, HSCs derive from a subset of aortic endothelial cells that undergo endothelial-to-hematopoietic transition to become HSCs. During this dynamic process, Gpr56 is the most upregulated receptor in emerging HSCs. The Gpr56 protein is a cell-surface receptor that is likely involved in the regulation of HSC function. However, its role in mammalian haematopoiesis remains unknown. In this study, we examine the function of Gpr56 during in vivo and in vitro HSC development. To explore the effect of Gpr56 loss-of-function, we characterize the functional role of mouse embryonic stem cell (ESC) derived hematopoietic stem and progenitor cells (HSPCs) and in vivo mouse HSCs that lack Gpr56. Gpr56-deficient HSPCs have a differentiation bias towards myeloid cells and the upregulation of another GPCR, Gpr97 appeared to compensate for Gpr56 deletion. The simultaneous deletion of Gpr56 and Gpr97 severely impaired the production of HSPCs, suggesting that both GPCRs are required in vitro for HSPC generation. Conversely, Gpr56 overexpression during human in vitro PSC differentiation resulted in an important HSPC expansion. In summary, these data demonstrate that the lack of Gpr56 alters the HSPC differentiation output, while its overexpression enhances human in vitro HSPCs generation. This work represents the most comprehensive and current study of Gpr56 in mammalian haematopoiesis and these findings will improve novel strategies to generate de novo HSC and/or promote their ex vivo expansion.