The cell fate decisions of haemopoietic stem cells (HSC) are controlled intrinsically and extrinsically by the bone marrow (BM) niche. Mature megakaryocytes (MM), responsible for platelet production, account for ~0.1-0.2% of BM cells and are randomly distributed within murine long bones. When Lin-Sca-1+Kit+CD150+CD48- HSC isolated from the endosteal region of murine long bones (eLSKSLAM) were co-cultured with SSChighCD41bright MM, we found that prospectively isolated MM were able to significantly increase the proliferation of eLSKSLAM (p<0.0001). Furthermore, the progeny maintained their haemopoietic transplant potential, giving rise to long-term multi-lineage reconstitution in vivo. When eLSKSLAM were cultured in MM conditioned media, increased eLSKSLAM proliferation was retained (p<0.001); demonstrating cell contact was not necessary. In addition, when culture media was supplemented with two factors known to be released by MM, insulin-like growth factor binding protein-3 (IGFBP-3) and insulin-like growth factor-1 (IGF-1), these factors worked synergistically to increase eLSKSLAM proliferation in a dose dependent manner; mimicking that seen in the presence of MM (p<0.001).We have also demonstrated that eLSKSLAM express the IGF-1 receptor and this increased eLSKSLAM proliferation was blocked by an anti-IGF-1 neutralising antibody (p<0.001). MM contain IGFBP-3 and IGF-1 transcripts and are also known to endocytose these factors. We recently developed a method for the prospective isolation of scavenging BM endothelial cells and identified them as an additional source of IGFBP-3. During MM maturation, DNA replication occurs without cytokinesis, resulting in polyploid cells up to 65μm in size. We have optimised a strategy for isolating MM based on ploidy and found that only when 8N, 16N and 32N MM were present in combination, and not in isolation, was there a significantly increase in eLSKSLAM proliferation. Our data suggests that MM are an important component of the HSC niche and play a role in the regulation and proliferation of HSC.