Human induced pluripotent stem cells (iPSCs) provide a highly consistent cell source for the ex-vivo production of therapeutic T cells.Of the different T cell subtypes, the CD8αβ+ T cell has the strongest clin. precedence in autologous and healthy-donor derived CAR-T cell products yet has historically been challenging to generate from iPSCs.Notch signaling is required to drive T-lineage differentiation and in conventional culture protocols is delivered by Delta-Like 4 (DLL4)-expressing feeder cells or DLL4 protein-coated tissue culture vessels.These methods have not yielded efficient production of CD8αβ+ T cells from iPSCs and lack control of ligand dose and timing.We have developed the Engineered Thymic Niche (ETN) platform, a fully defined custom reagent consisting of magnetic beads coated with DLL4 and vascular cell adhesion mol. 1 (VCAM-1).The ETN allows for both temporal and intensity modulation of Notch signalling by adding or removing beads from culture vessels and by increasing or decreasing the surface concentration of DLL4 and VCAM-1 on the beads.To characterize the ETN, we developed assays for bead size, concentration, protein content and function.Specifically, on-bead DLL4 content was quantified using an immunofluorescence-based Notch-1 binding capacity assay, while the bead function was assessed by measuring Notch responsive gene expression and cell phenotype in response to ETN dose during differentiation of iPSC-derived CD34+ cells.We demonstrated that increasing both bead concentration and protein d. (10-fold range) in cultures initiated with CD34+ HSPC cells, modulates the expression of Notch responsive genes (Notch1, TCF7, DTX1, BCL11B, HES1).Higher gene expression correlated to increased expression of ProT markers (20 to 60%) after 14 days.Finally, we show that ETN is remarkably stable, with ongoing shelf-life study showing > 9 mo stability at 4 °C.Furthermore, the ETN is stable for 35 days at 37 °C and can withstand freezing as assessed by size distribution and Notch-1 binding.Looking ahead, the aseptic, scalable ETN manufacturing process will incorporate qualified raw materials, including GMP-level DL4 and VCAM.Our advances in ETN bead design and characterization demonstrate that we have a stable reagent that enables the in-vitro control of Notch signaling required for consistent differentiation of iPSC derived CD34+ cells to mature T cells, opening a path to clin. translation of ETN for cell production