CD38 is a surface receptor able to induce activation, proliferation, and survival of human and mouse lymphocytes; this molecule is expressed on the surface of both mature and immature B cells. In this work, the function of CD38 in the maturation of murine B lymphocytes in the spleen was analyzed. The results showed that CD38 is highly expressed on Transitional 2 (T2) B lymphocytes with an intermediate expression on Transitional 1 (T1) and mature follicular B cells (M). Correlating with a high expression of CD38, T2 cells are also larger and more granular than T1 or M B cells. T2 cells also showed high levels of other molecules, which indicate an activated phenotype. CD38 crosslinking induced proliferation and maturation of T2 B lymphocytes; in contrast, T1 subset died by apoptosis. Finally, CD38 stimulation of T2 B lymphocytes obtained from Btk-, Lyn-, or Fyn-deficient mice showed a defective differentiation; similarly, drugs interfering with PI3K or ERK decreased the proliferation or differentiation of this subset. This suggests that these molecules participate in the CD38 signaling pathway. As a whole, the results indicate that CD38 plays an important role in the regulation of B-cell maturation in the spleen.