Abstract Developing B cells in mutant ali/ali rabbits predominantly utilize VH4 during VDJ gene rearrangement. However, most of the VH4-utilizing B cells encounter a severe developmental block at the pre-B cell stage. In contrast, wildtype rabbit B cells predominantly rearrange VH1, a gene >90% identical to VH4, and develop normally. We seek to determine how these nearly identical VH genes have profoundly different effects on B cell development. We hypothesize that VH4-utilizing cells are blocked at the pre-B stage due to weak interaction of the μ-chain with surrogate light chain (SL). We expressed VH4-utilizing μ-chains in a pro-B cell line that expresses SL and found that VH4-encoded μ-chains exhibit a 2 fold reduction in preBCR surface localization compared to control VH1-encoded μ-chains. Remarkably, a single amino acid substitution in VH4-framework region 2 (FR2) from tyrosine to an evolutionarily conserved tryptophan, found in VH1 and most VH genes of other species, restored surface preBCR expression. We are testing if this tryptophan substitution in FR2 of VH4 is sufficient to rescue pre-B cell development. These studies should identify evolutionarily conserved VH structural regions required for efficient preBCR formation and B cell development.