A whole-genome CRISPR/Cas9 screen identified ATP2A2, the gene encoding the Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2 protein, as being important for V(D)J recombination. SERCAs are ER transmembrane proteins that pump Ca2+ from the cytosol into the ER lumen to maintain the ER Ca2+ reservoir and regulate cytosolic Ca2+-dependent processes. In preB cells, loss of SERCA2 leads to reduced V(D)J recombination kinetics due to diminished RAG-mediated DNA cleavage. SERCA2 deficiency in B cells leads to increased expression of SERCA3, and combined loss of SERCA2 and SERCA3 results in decreased ER Ca2+ levels, increased cytosolic Ca2+ levels, reduction in RAG1 and RAG2 gene expression, and a profound block in V(D)J recombination. Mice with B cells deficient in SERCA2 and humans with Darier disease, caused by heterozygous ATP2A2 mutations, have reduced numbers of mature B cells. We conclude that SERCA proteins modulate intracellular Ca2+ levels to regulate RAG1 and RAG2 gene expression and V(D)J recombination and that defects in SERCA functions cause lymphopenia.