Current in vitro human erythroid culture platforms require abnormally high cytokine supplementation and use lower cell density ( 107/mL) when perfused with cytokine-free media in long-term culture. In order to study the role of this manufactured HFBR microenvironment on spatiotemporal physiologic erythropoiesis, we now extend our previous reports by implementing 5-fold less cytokine concentrations than those used in typical ex vivo erythropoietic cultures. Herein, we show a >107 red cell harvest from the HFBR culture over 28-days with spontaneous expansion of stromal cells, maintenance of erythroid progenitor pools, and formation of stromal and erythroid cell niches in defined areas within the HFBR structure with differential in situ production of 23 growth factors varying over time. The 5.25 mL HFBR scaffold was inoculated with 108 CBMNCs and HFs were rapidly perfused (20 mL/h) with serum-free StemSpan medium gradually supplemented with a cytokine gradient of decreasing SCF (50 - 0 ng/mL) and increasing EPO (0 - 0.3 U/mL) over 28 days in order to maintain progenitor cells whilst inducing erythropoiesis. Quantitative confocal microscopy analyses of HFBR sections demonstrated that DAPI+ CBMNCs maintained high cell density (>107/mL), and high viability (>80%), while more than 107 enucleated cells were filtered through HFs over the 28-day culture. Inside the HFBR, hematopoietic progenitor cells were maintained (total of 3.1∙106 CD34+ and 5.5∙106 CKIT+ MNCs) while erythroid cells were expanded across various stages of maturation (28-day total increase of 1.2∙107 EPOR+, 1.8∙107 CD71+, and 2.3∙107 CD235a+ MNCs); CD235a+mature red cell phenotypes were enriched 10-fold in the HF filtrate over 28 days. Stromal cells expanded and differentiated during the 28-day HFBR culture with a total increase of mesenchymal stem cell marker Stro-1 (2.2∙107 cells), pre-osteoblast marker osterix (OSx; 1.6∙107 cells), and mature osteoblast marker osteopontin (OPN; 0.5∙107 cells). Expression of human collagen-1, fibronectin, and laminin-2 was detected by microscopy, while enzyme-linked immunoassays on HFBR filtrate detected 23 multilineal, unsupplemented cytokine profiles including interleukins produced primarily from day 0-12 (IL-6, IL-10, IL-21) as well as colony stimulating factors and stromal growth factors which increased in production from day 20-28 (G-CSF, GM-CSF, EGF, VEGF, Ang-2, PDGF, FGF-β). Using a novel confocal microscopy computational analyses that we have developed, DAPI+MNCs were found to self-associate into expanding 50-500µm clusters throughout the 28-day culture which increased local cell density 10-20 fold, representing niche-like areas. At day 14 and 28, MNCs formed clustered niches far from HFs which expressed hypoxic (HIF1a, PIMO), stromal, and erythroid markers (Stro-1, OSx, collagen-1, laminin-2, VCAM-1, CD45, EPOR: >1400µm from HFs). At day 28, 3-fold more MNC clusters formed near HFs and were comprised of hematopoietic progenitor and erythroid phenotypes (CD45, CD34, CKIT, CD235a, CD71: Our data suggested that the dense inoculation of CBMNCs in a serum-free HFBR platform using physiologic concentrations of SCF and EPO enabled the long-term simultaneous differentiation of human erythroid, stromal, and osteogenic lineages, and the generation of an ex vivo erythroid inductive environment. This environment maintained multilineal progenitors, enabled harvest of mature erythrocytes, generated cytokine support in situ, and formed interactive cell niches which could be quantitatively mapped in spatiotemporal zones. The HFBR we have developed may represent a more physiologically-relevant culture system to study ex vivo erythropoiesis and could potentially provide a platform for translational cell expansion protocols. Disclosures No relevant conflicts of interest to declare.