Haematopoietic stem cell (HSC) transplantation is a key therapy for patients with leukaemia or bone marrow failure. Repopulating HSCs derived from human pluripotent stem cells (hPSCs) would provide a source of blood stem cells for many patients who might benefit from this therapy but lack a suitable donor.Success is contingent on the development of hPSC differentiation protocols that generate and support the developmental continuum from correctly patterned haemogenic endothelium to the emerging preHSCs, and their subsequent maturation to repopulating (r) HSCs. Whilst many hPSC differentiation protocols generate blood cells similar to those arising from the human yolk sac, our recent work has demonstrated that we can differentiate blood cells from hPSCs that phenotypically and transcriptionally resemble the preHSCs found in the aorta-gonad-mesonephros (AGM) of the human embryo. An important finding from these studies was that the expression of HOXA genes distinguished yolk sac like (HOXA-) from AGM-like (HOXA+) endothelium and blood cells. In response to these observations, we developed a hPSC reporter line in which tdTOMATO is linked to expression of HOXA5, a gene that is expressed in intra-embryonic haemogenic endothelium, preHSCs and rHSCs. Validation of the reporter demonstrated that HOXA5-expressing cells were also enriched for expression of the whole HOXA cluster. We have shown that this line enables us to enumerate the proportion of endothelia and blood cells that express HOXA5 during differentiation, and that this faithfully reflects the bias towards a yolk sac (TOMATO-) or AGM-like (TOMATO+) fate imposed by the protocol used. This line will enable fine-tuning of the differentiation process to identify conditions that support development and maintenance of cells that carry a stem cell 'signature' and also express HOXA genes – a hypothesized requirement for rHSC.