Ankyrin repeats are well-known structural modules that mediate interactions between a wide spectrum of proteins. The regulatory factor X with ankyrin repeats (RFXANK) is a subunit of a tripartite RFX complex that assembles on promoters of major histocompatibility complex class II (MHC II) genes. Although it is known that RFXANK plays a central role in the nucleation of RFX, it was not clear how its ankyrin repeats mediate the interactions within the complex and with other proteins. To answer this question, we modeled the RFXANK protein and determined the variable residues of the ankyrin repeats that should contact other proteins. Site-directed alanine mutagenesis of these residues together with in vitro and in vivo binding studies elucidated how RFXAP and CIITA, which simultaneously interact with RFXANK in vivo, bind to two opposite faces of its ankyrin repeats. Moreover, the binding of RFXAP requires two separate surfaces on RFXANK. One of them, which is located in the ankyrin groove, is severely affected in the FZA patient with the bare lymphocyte syndrome. This genetic disease blocks the expression of MHC II molecules on the surface of B cells. By pinpointing the interacting residues of the ankyrin repeats of RFXANK, the mechanism of this subtype of severe combined immunodeficiency was revealed.