B cell development and Ig rearrangement are governed by cell type- and developmental stage-specific transcription factors. PU.1 and Spi-B are E26-transformation-specific transcription factors that are critical for B cell differentiation. To determine whether PU.1 and Spi-B are required for B cell development in the bone marrow, Spi1 (encoding PU.1) was conditionally deleted in B cells by Cre recombinase under control of the Mb1 gene in Spib (encoding Spi-B)-deficient mice. Combined deletion of Spi1 and Spib resulted in a lack of mature B cells in the spleen and a block in B cell development in the bone marrow at the small pre-B cell stage. To determine target genes of PU.1 that could explain this block, we applied a gain-of-function approach using a PU.1/Spi-B- deficient pro-B cell line in which PU.1 can be induced by doxycycline. PU.1-induced genes were identified by integration of chromatin immunoprecipitation-sequencing and RNA-sequencing data. We found that PU.1 interacted with multiple sites in the Igk locus, including Vk promoters and regions located downstream of Vk second exons. Induction of PU.1 induced Igk transcription and rearrangement. Upregulation of Igk transcription was impaired in small pre-B cells from PU.1/Spi-B-deficient bone marrow. These studies reveal an important role for PU.1 in the regulation of Igk transcription and rearrangement and a requirement for PU.1 and Spi-B in B cell development.