Chromosomal interactions regulate genome functions, such as transcription, via dynamic chromosomal organization in the nucleus. In this study, we attempted to identify genomic regions that physically bind to the promoter region of the Pax5 gene, which encodes a master regulator for B cell lineage commitment, in a chicken B cell line, DT40, with the goal of obtaining mechanistic insight into transcriptional regulation through chromosomal interaction. We found that the Pax5 promoter bound to multiple genomic regions using locus-specific chromatin immunoprecipitation (locus-specific ChIP), a method for locus-specific isolation of target genomic regions, in combination with next-generation sequencing (NGS). Comparing chromosomal interactions in wild-type DT40 with those in a macrophage-like counterpart, we found that some of the identified chromosomal interactions were organized in a B cell-specific manner. In addition, deletion of a B cell-specific interacting genomic region in chromosome 11, which was marked by active enhancer histone modifications, resulted in moderate but significant down-regulation of Pax5 transcription. Together, these results suggested that Pax5 transcription in DT40 is regulated by B cell-specific inter-chromosomal interactions. Moreover, these analyses showed that locus-specific ChIP combined with NGS analysis is useful for non-biased identification of functional genomic regions that physically interact with a locus of interest.