B cell development is characterized by marked changes in Ig repertoire, which include shifts in the pattern of V(H) segment usage. B cell precursors characteristically utilize a restricted set of V(H) segments, while mature B cell populations use a wide range of V(H) segments. V(H)81x is an example of a V(H) segment that is highly utilized in B cell precursors, but is rarely utilized in mature B cells. To dissect the molecular and cellular requirements for Ig repertoire maturation, we have examined V(H)81x usage in an in vitro model of B cell development. We find that primary fetal or adult B cell progenitors differentiating in vitro mimic progenitors differentiating in vivo with respect to V(H)81x overusage and subsequent decline in V(H)81x usage, showing that neither of these events is dependent on the intact architecture of the primary lymphoid organ or contact with stromal cells. The relative decline in V(H)81x usage in cultures initiated with adult progenitors was associated with a decrease in the ratio of productive/nonproductive V(H)81x-DJ(H) rearrangements; however, an increase in this ratio was observed in identical cultures initiated with fetal progenitors. This result indicates a difference in selection of V(H)81x-encoded heavy chains that is intrinsic to fetal and adult B cell progenitors. Thus, while the relative decline in V(H)81x usage during adult development can be at least partially explained by selection against cells bearing V(H)81x-encoded heavy chains, other mechanisms must be postulated to explain the decline in V(H)81x usage during fetal development.