Children with Down's syndrome (DS) have 20–50-fold higher incidence of all leukaemias (lymphoid and myeloid), for reasons not understood. As incidence of many solid tumours is much lower in DS, we speculated that disturbed early haematopoietic differentiation could be the cause of increased leukaemia risk. If a common mechanism is behind the risk of both major leukaemia types, it would have to arise before the bifurcation to myeloid and lymphoid lineages. Using the transchromosomic system (mouse embryonic stem cells (ESCs)) bearing an extra human chromosome 21 (HSA21)) we analyzed the early stages of haematopoietic commitment (mesodermal colony formation) in vitro. We observed that trisomy 21 (T21) causes increased production of haemogenic endothelial cells, haematopoietic stem cell precursors and increased colony forming potential, with significantly increased immature progenitors. Transchromosomic colonies showed increased expression of Gata-2, c-Kit and Tie-2. A panel of partial T21 ESCs allowed us to assign these effects to HSA21 sub-regions, mapped by 3.5 kbp-resolution tiling arrays. The Gata-2 increase on one side, and c-Kit and Tie-2 increases on the other, could be attributed to two different, non-overlapping HSA21 regions. Using human-specific small interfering RNA silencing, we could demonstrate that an extra copy of RUNX1, but not ETS-2 or ERG, causes an increase in Tie-2/c-Kit levels. Finally, we detected significantly increased levels of RUNX1, C-KIT and PU. 1 in human foetal livers with T21. We conclude that overdose of more than one HSA21 gene contributes to the disturbance of early haematopoiesis in DS, and that one of the contributors is RUNX1. As the observed T21-driven hyperproduction of multipotential immature precursors precedes the bifurcation to lymphoid and myeloid lineages, we speculate that this could create conditions of increased chance for acquisition of pre-leukaemogenic rearrangements/mutations in both lymphoid and myeloid lineages during foetal haematopoiesis, contributing to the increased risk of both leukaemia types in DS.