Factors responsible for long-term survival and proliferation of human hematopoietic stem cells (hHSC), and their mechanisms of action, remain to be defined. We previously showed that specific O-sulfated heparan sulfate (OS-HS) improves human long-term culture initiating cell (LTC-IC) maintenance for up to 5 wks in vitro. This is related to the ability of OS-HS to bind to and modulate the activity of heparin-binding cytokines on primitive hematopoietic progenitors (PHP). Recent studies indicate that bone morphogenetic proteins (BMPs) influence the development of embryonic hematopoiesis and also augment short-term survival and proliferation of hHSC. Since HS may modulate BMP activity, we examined how combinations of OS-HS, BMPs and specific BMP antagonists influence PHP in umbilical cord blood (UCB). First, we confirmed by real-time quantitative RT-PCR (qRT-PCR) that UCB CD34+ and/or CD34+/CD38− cells (using linear mRNA amplification) constitutively express transcripts for BMP-4 and its inhibitor Chordin, BMP receptors BMPR-IA, BMPR-IB, BMPR-II, AcvR-II and AcvR-IIB, downstream signaling proteins SMAD-1 and -5, and target genes upregulated by BMPs including Inhibitors of DNA binding (Id) proteins 1–4, and determined their relative levels of expression. BMP-4 upregulated Id2 expression 17-fold, confirming that the BMP signaling pathway is functionally active in CD34+ cells. Next, we demonstrated that OS-HS may protect BMP-4 from its inhibitors, using Western immunoblotting of immunoprecipitated proteins to show that direct binding of the antagonist Chordin to BMP-4 is inhibited by OS-HS in a dose-dependent manner. Finally, we examined the effect of exogenous supplementation with 6 BMPs, or inhibition of endogenous BMPs by 7 antagonists, on short-term (2 wk) and long-term (5 wk) LTC-IC maintenance in UCB CD34+/CD38− cells cultured in presence of OS-HS, Flt3-ligand and thrombopoietin. Long-term LTC-IC maintenance was enhanced by BMP-4 (LTC-IC maintenance: 164 +/− 11% compared to culture without BMP-4; P=0.0001) but reduced by BMP-2 or BMP-7 (P<0.005), whereas BMP-3 had no effect. In combination, BMP-3, -4 and -7 offset each other, with no net effect on LTC-IC maintenance. The simultaneous presence of BMPs 2–7 was markedly inhibitory to both short-term (56 +/− 11%; P=0.0018) and long-term (38 +/− 11%; P=0.0024) LTC-IC maintenance. Inhibition of endogenous BMPs by Noggin (which antagonizes BMP-2, -4 and -7 and GDF-5) most effectively enhanced long-term LTC-IC maintenance (320 +/− 36%; P=0.0002). In contrast, other antagonists that inhibit different groups of BMPs did not enhance long-term LTC-IC maintenance. These results indicate that (a) the BMP signaling pathway is expressed and functionally active in human PHP, (b) different members of the BMP family have distinct stimulatory or inhibitory effects on long-term LTC-IC maintenance, (c) endogenously expressed BMPs are extremely important modulators of long-term PHP maintenance, and (d) the ability of OS-HS to augment long-term in vitro PHP maintenance may be related to its effect on endogenous BMP activity. Our data suggest that selective modulation of BMP signaling may be essential for achieving long-term in vitro maintenance of human PHP. Ongoing studies are investigating how OS-HS affects BMP signaling, and the downstream effects of these molecules on target genes that influence stem cell decisions and fate.