BM-derived MSCs efficiently support the generation of KIR-expressing NK cells from early hematopoietic progenitors. CD34⁺ HSPCs were isolated from CB and seeded at 3 × 10³ cells per well on monolayers of the indicated stromal cell types (MSC, USSC) or fibroblasts (STF5). Cell number (A) and expression of CD56 (B) were measured weekly during culture by flow cytometry. Maximum expression frequency of NKG2A (C) and KIR (D) was monitored for each of the indicated cell lines using a mixture of KIR-specific monoclonal antibodies for inhibitory KIR2D and KIR3D receptors (data are mean frequency and standard deviation of 3 independent experiments). (E) Flow cytometric analysis of cultures at day 14 (top panel), day 21 (middle panel), and day 28 (bottom panel). Cells were gated on the CD56⁺ subset, and expression of the NK cell receptors KIR2DL1, KIR2DL3, KIR3DL1, and NKG2A was analyzed.