Haematopoietic stem cell progenitor cells (HSPCs) have substantial research and therapeutic value, but the in vitro production of HSPCs from pluripotent stem cells remains elusive. A novel 3D culture method, known as a gastruloid, can mimic gastrulation in embryogenesis, which is crucial to the emergence of HSPC precursors, highlighting the potential to develop this protocol as an in vitro platform for haematopoietic research. This project aims to adapt the previously published gastruloid culture protocol and develop it into a haemogenic gastruloid protocol to produce HSPCs from mouse ES cells and model haematopoiesis which occurs at the aorta-gonad-mesonephros (AGM) region of the mouse embryo. In this project, the gastruloid culture protocol is modified to maintain a gastruloid (gastrulation organoid) for up to 216 hr by introducing a 2iLIF pretreatment and a panel of cytokines throughout the culture. Under the new culture conditions, gastruloids can generate haemogenic endothelium and pro- and pre-HSCs in sequential order, as detected by a combination of markers in flow cytometry assays (Flk-1, c-Kit, CD41 and CD45). Concomitant use of cytokines VEGF and FGF2 can facilitate the emergence of cells with the property of haemogenic endothelial cells, pro-HSCs and type 1 pre-HSCs from 96 to 144 hr. Applying Shh, Flt-3l, TPO, and SCF accordingly can promote the formation CD45+ cells, which is indicative of the formation of type 2 pre-HSCs at 192 and 216 hr. The emergence of these haematopoietic makers suggests stepwise haematopoiesis is recapitulated in the haemogenic gastruloid. The AGM-like haematopoietic clusters are observed at 192 and 216 hr under confocal imaging, further suggesting that definitive haematopoiesis likely occurs in the gastruloid under the optimised protocol. The CFC assay shows that gastruloid cells have the potential to form multipotential myeloid and lymphoid haematopoietic progenitors. The CD45+ cells from the 216 hr gastruloids can occasionally be enriched in OP9 co-culture, and they can also sometimes form committed B-cell linage progenitors with OP9 cells. The chick embryo chorioallantoic membrane (CAM) assay and mouse transplantation experiments imply that the haemogenic gastruloid cells may possess engraftment capability. Single cell SMART sequencing data confirms that gastruloids have haematopoietic gene expression profiles similar to the AGM region of the mouse embryo and captures successive progenitors with erythroid, myeloid-lymphoid and HSC-like signatures. Finally, this haemogenic gastruloid protocol has been applied to other mouse ES cell lines to generate gastruloids, and they can also recapitulate stepwise haematopoiesis. This study has successfully developed a haemogenic gastruloid protocol with a cocktail of haematopoietic cytokines. This haemogenic gastruloid is able to form AGM-like haematopoietic clusters and recapitulate stepwise definitive haematopoiesis to produce haematopoietic progenitors. In the future, this protocol could be developed to validate and reduce the use of animals in scientific research. The results from this study could also be useful preliminary data for developing a human haemogenic gastruloid protocol. Finally, this protocol could be applied as a drug screening or disease modelling tool, such as an infant leukaemia disease model.