Unmethylated CpG DNA activation of naive CD27⁻ B cells has been reported to require B-cell–receptor (BCR) cross-linking. We describe a culture system using CpG DNA with sequential steps for T-cell–independent activation of naive CD19⁺CD27⁻ human peripheral blood B cells that induces efficient CD138⁺ plasma-cell differentiation. CD27⁺ and CD27⁻ B cells were cultured in a 3-step system: (1) days 0 to 4: CpG, IL-2/10/15; (2) days 4 to 7: IL-2/6/10/15 and anti-CD40L; (3) days 7 to 10: IL-6/15, IFN-α, hepatocyte growth factor, and hyaluronic acid. Both CD27⁺ and CD27⁻ B cells up-regulated intracytoplasmic TLR-9 following CpG DNA activation. CD27⁻ B-cell activation required cell-cell contact. Both naive and memory B cells progressed to a plasma-cell phenotype: CD19^lowCD20^lowCD27⁺CD38⁺HLA-DR^low. Seventy percent of the CD27⁻-derived CD138⁺ cells demonstrated productive V chain rearrangements without somatic mutations, confirming their origin from naive precursors. Plasma cells derived from CD27⁺ B cells were primarily IgG⁺, while those from CD27⁻ B cells were IgM⁺. Our results indicate that under certain conditions, naive B cells increase TLR-9 expression and proliferate to CpG DNA stimulation without BCR signaling. In addition to its immunologic significance, this system should be a valuable method to interrogate the antigenic specificity of naive B cells.