Transitional cells represent a crucial step in the differentiation and selection of the mature B cell compartment. Human transitional B cells have previously been variably identified based on the high level of expression of CD10, CD24, and CD38 relative to mature B cell populations and are expanded in the peripheral blood following rituximab-induced B cell-depletion at reconstitution. In this study, we take advantage of the gradual acquisition of the ABCB1 transporter during B cell maturation to delineate refined subsets of transitional B cells, including a late transitional B cell subset with a phenotype intermediate between T2 and mature naive. This late transitional subset appears temporally following the T1 and T2 populations in the peripheral compartment after rituximab-induced B cell reconstitution (and is thus termed T3) and is more abundant in normal peripheral blood than T1 and T2 cells. The identity of this subset as a developmental intermediate between early transitional and mature naive B cells was further supported by its ability to differentiate to naive during in vitro culture. Later transitional B cells, including T2 and T3, are found at comparatively increased frequencies in cord blood and spleen but were relatively rare in bone marrow. Additional studies demonstrate that transitional B cells mature across a developmental continuum with gradual up-regulation of mature markers, concomitant loss of immature markers, and increased responsiveness to BCR cross-linking in terms of proliferation, calcium flux, and survival. The characterization of multiple transitional B cell subpopulations provides important insights into human B cell development.