2016 © The Authors. Developmental checkpoints eliminate B cells that synthesize defective immunoglobulin (Ig) heavy (HC) and light (LC) chains. The first checkpoint tests mHCs paired with VpreB/l5 in a pre-B cell receptor (pre-BCR) to determine whether the mHC will be able to bind conventional LCs to form membrane IgM. VpreB and l5 also create a sensing site that interacts with the mHC antigen-binding region complementarity-determining region (CDR)–H3; however, whether this site contributes to Ig repertoire selection and function is unknown. We analyzed the amino acid content of CDR-H3s from HCs cloned from living and apoptotic pre-B cells and from IgG-antigen structures. Using a panel of DH gene–targeted mice, we showed that progressively reducing CDR-H3 tyrosine content increasingly impaired pre-BCR checkpoint passage. Counting from cysteine at framework 3 position 96, we found that VpreB particularly selected for tyrosine at CDR-H3 position 101 and that Y101 also bound antigen in IgG-antigen structures. Therefore, in addition to its stabilization role in the pre-BCR, VpreB also acts as an early invariant antigen by selecting for particular CDR-H3 amino acids. These interactions shape the specificity of the IgG humoral response and may thus impose limitations on development of certain neutralizing antibodies.