Progeny of AFT024 cultures give rise to NK cells and CD10⁺/CD19⁺ B-lineage cells. CD34⁺/Lin⁻/DR⁻ cells were cultured in limiting dilutions on AFT024 with IL-7, FL, KL, and IL-2 added at culture initiation. Media was half changed weekly with fresh media supplemented with IL-2 alone. Individual wells were harvested and analyzed by flow cytometry after three-color staining (CD56-PE, CD10-FITC, CD19-PerCP) using appropriate isotype controls. The representative example shown is from a culture well initiated with 130 cells. (A) Shows the two-color analysis of CD56 and CD19. CD19 positive cells were then backgated onto the forward and side scatter plot (B). The CD19⁺ B-lineage cells (black dots) were significantly smaller than NK cells (medium gray) found within the lymphoid window of the same culture. Backgating to find B-lineage cells in this very small lymphocyte gate (R2) was used as an absolute criteria for all cultures determined positive for B-lineage cells. (C) Shows a representative example of the CD10⁺/CD19⁺ B-lineage cells based on the R2 gate.