CD27, a member of the TNFR superfamily, is used to identify human memory B cells. Nonetheless, CD27⁺ B cells are present in patients with HIGM1 syndrome who are unable to generate GCs or memory B cells. CD27⁺IgD⁺ fetal B cells are present in umbilical cord blood, and CD27 may also be a marker of the human B1-like B cells. To define the origin of naïve CD27⁺IgD⁺ human B cells, we studied B cell development in both fetal and adult tissues. In human FL, most CD19⁺ cells coexpressed CD10, a marker of human developing B cells. Some CD19⁺CD10⁺ B cells expressed CD27, and these fetal CD27⁺ cells were present in the pro-B, pre-B, and immature/transitional B cell compartments. Lower frequencies of phenotypically identical cells were also identified in adult BM. CD27⁺ pro-B, pre-B, and immature/transitional B cells expressed recombination activating gene-1, terminal deoxynucleotidyl transferase and Vpre-B mRNA comparably to their CD27⁻ counterparts. CD27⁺ and CD27⁻ developing B cells showed similar Ig heavy chain gene usage with low levels of mutations, suggesting that CD27⁺ developing B cells are distinct from mutated memory B cells. Despite these similarities, CD27⁺ developing B cells differed from CD27⁻ developing B cells by their increased expression of LIN28B, a transcription factor associated with the fetal lymphoid lineages of mice. Furthermore, CD27⁺ pro-B cells efficiently generated IgM⁺IgD⁺ immature/transitional B cells in vitro. Our observations suggest that CD27 expression during B cell development identifies a physiologic state or lineage for human B cell development distinct from the memory B cell compartment.