To create antibody diversity, B lymphocyte development is characterized by the ordered rearrangement of first immunoglobulin (Ig) heavy chain gene segments and then Ig light-chain gene segments. Early in B-cell development, expression of a pre-B-cell receptor (pre-BCR) composed of membrane-bound Ig heavy chain protein associated with surrogate light-chain (SLC) proteins serves as a critical checkpoint that monitors for functional heavy chain rearrangement. Signaling from the pre-BCR induces clonal expansion, but it also turns off transcription of the genes for the SLC proteins lambda5 and VpreB, which limits this proliferation. Here we show that signaling from the pre-BCR rapidly down-regulates lambda5 and VpreB and also the co-receptor CD19 in primary pre-B-cells. We show that calcium (Ca(2+)) signaling is essential for this silencing of the SLC and CD19 genes. The SLC genes are activated by the E2A transcription factor, and we show that E2A is required for pre-BCR-mediated regulation of the genes. E2A mutated in its binding site for the Ca(2+) sensor protein calmodulin, and thus with calmodulin-resistant DNA binding, makes lambda5, VpreB and CD19 expression resistant to the inhibition following pre-BCR activation. Thus, Ca(2+) down-regulates SLC and CD19 gene expression upon pre-BCR activation through inhibition of E2A by Ca(2+)/calmodulin.