Abstract NK cells are potent cellular immunotherapeutic agents against a wide array of human malignancies. Ex vivo expansion of NK cells to achieve clinically relevant numbers must overcome poor in vitro growth kinetics, low starting percentages within the mononuclear cell fraction (especially from autologous donors with active disease), and limited in vivo life span. We targeted three universally critical NK signaling pathways, namely IL-21, 4-1BB, and SLAM family member 4 (SLAMF4), to increase NK cell proliferation and enhance survival. We genetically-engineered HLA-A-ve and -B-ve K562 cells to enforce expression of membrane-bound IL-21 (mbIL21), 4-1BB-L, and CD48, forming a universal antigen presenting cell (UAPC) to generate highly potent clinical-grade umbilical cord blood (CB-NK) or peripheral blood NK cells (PB-NK). While the mbIL21 and 4-1BB signaling nexuses have been utilized previously, we highlight here SLAMF-mediated immunological sculpting of NK cells for clinical applications. SLAMF triggering of co-receptors modulate NK cell activation, in particular through high-avidity interactions between SLAMF4 (2B4/CD244) and its heterophilic, robust affinity, and physiological ligand CD48, a glycosyl-phosphatidyl-inositol (GPI)-anchored cell surface protein. Upon ligand binding and receptor phosphorylation, SLAMF4 recruits PTPN11/SHP-2 and SH2D1A/SAP for downstream signaling, including significant increases in NK cell-mediated cytotoxicity, granule exocytosis, and production of IFN-γ and IL-2. Other than a subset of low surface density expressers from aging subjects consistently associated with inefficient and impaired activating signal transduction, the majority of NK cells express SLAMF4. The functional prominence of SLAMF4, presently recognized as an activating co-receptor, is also evidenced by loss-of-function mutations associated with X-linked lymphoproliferative (XLP) disease. We achieved log-scale expansion of NK cells with UAPC (>1000 fold in 2 weeks), with excellent purity (>99% CD56+ve/CD3−ve and < 1% CD3+ve cells), without indications of senescence/exhaustion, even after 4 weeks of culture. Surface molecular phenotypes of UAPC-expanded CB-NK cells exhibited a phenotype similar to CB-NK cells expanded without SLAMF4. Synergistic signals from IL-21/STAT3, 4-1BB/4-1BBL, and SLAMF4 drive tonic NK propagation, supporting their clinical application. Our novel and clinically accessible platform technology for generation of high purity CB-NKs, a promising source of fresh and cryopreserved allogeneic NK cells, is well-suited for adoptive cancer immunotherapy. Disclosures Champlin: Sanofi: Research Funding; Otsuka: Research Funding. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.