The cellular origin of chronic lymphocytic leukemia (CLL) is still debated, although this information is critical to understanding its pathogenesis. Transcriptome analyses of CLL and the main normal B cell subsets from human blood and spleen revealed that immunoglobulin variable region (IgV) gene unmutated CLL derives from unmutated mature CD5⁺ B cells and mutated CLL derives from a distinct, previously unrecognized CD5⁺CD27⁺ post–germinal center B cell subset. Stereotyped V gene rearrangements are enriched among CD5⁺ B cells, providing independent evidence for a CD5⁺ B cell derivation of CLL. Notably, these CD5⁺ B cell populations include oligoclonal expansions already found in young healthy adults, putatively representing an early phase in CLL development before the CLL precursor lesion monoclonal B cell lymphocytosis. Finally, we identified deregulated proteins, including EBF1 and KLF transcription factors, that were not detected in previous comparisons of CLL and conventional B cells.