Substantial evidence indicates that signaling through the CD40 receptor (CD40) is required for germinal center (GC) and memory B-cell formation. However, it is not fully understood at which stages of B-cell development the CD40 pathway is activated in vivo. To address this question, we induced CD40 signaling in human transformed GC B cells in vitro and identified a CD40 gene expression signature by DNA microarray analysis. This signature was then investigated in the gene expression profiles of normal B cells and found in pre- and post-GC B cells (naive and memory) but, surprisingly, not in GC B cells. This finding was validated in lymphoid tissues by showing that the nuclear factor-κB (NF-κB) transcription factors, which translocate to the nucleus upon CD40 stimulation, are retained in the cytoplasm in most GC B cells, indicating the absence of CD40 signaling. Nevertheless, a subset of centrocytes and B cells in the subepithelium showed nuclear staining of multiple NF-κB subunits, suggesting that a fraction of naive and memory B cells may be subject to CD40 signaling or to other signals that activate NF-κB. Together, these results show that GC expansion occurs in the absence of CD40 signaling, which may act only in the initial and final stages of the GC reaction. (Blood. 2004;104: 4088-4096)