The intronic enhancer (E mu) of the immunoglobulin heavy chain (IgH) locus is critical for V region gene assembly. To determine E mu's subsequent functions, we created an Igh allele with assembled V(H) gene but with E mu removed. In mice homozygous for this E mu-deficient allele, B cell development was normal and indistinguishable from that of mice with the same V(H) knockin and E mu intact. In mice heterozygous for the E mu-deficient allele, however, allelic exclusion was severely compromised. Surprisingly, this was not a result of reduced suppression of V-DJ assembly on the second allele. Rather, the striking breakdown in allelic exclusion took place at the pre-B to immature B cell transition. These findings reveal both an important role for E mu in influencing the fate of newly arising B cells and a second checkpoint for allelic exclusion.