Endothelial and mesenchymal stromal cells (ECs/MSCs) are crucial components of hematopoietic bone marrow stem cell niches. Both cell types appear to be required to support the maintenance and expansion of multipotent hematopoietic cells, i.e. hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). With the aim to exploit niche cell properties for experimental and potential clinical applications, we analyzed the potential of primary ECs alone and in combination with MSCs to support the ex vivo expansion/maintenance of human hematopoietic stem and progenitor cells (HSPCs). Even though a massive expansion of total CD34⁺ HSPCs was observed, none of the tested culture conditions supported the expansion or maintenance of multipotent HSPCs. Instead, mainly lympho-myeloid primed progenitors (LMPPs) were expanded. Similarly, following transplantation into immunocompromised mice the percentage of multipotent HSPCs within the engrafted HSPC population was significantly decreased compared to the original graft. Consistent with the in vitro findings, a bias towards lympho-myeloid lineage potentials was observed. In our conditions, neither classical co-cultures of HSPCs with primary ECs or MSCs, even in combination, nor the xenograft environment in immunocompromised mice efficiently support the expansion of multipotent HSPCs. Instead, enhanced expansion and a consistent bias towards lympho-myeloid committed LMPPs were observed.