The Nuclear Factor I (NFI) family of site-specific DNA-binding proteins (also known as CTF or CAAT box transcription factor) functions both in viral DNA replication and in the regulation of gene expression. The classes of genes whose expression is modulated by NFI include those that are ubiquitously expressed, as well as those that are hormonally, nutritionally, and developmentally regulated. The NFI family is composed of four members in vertebrates (NFI-A, NFI-B, NFI-C and NFI-X), and the four NFI genes are expressed in unique, but overlapping, patterns during mouse embryogenesis and in the adult. Transcripts of each NFI gene are differentially spliced, yielding as many as nine distinct proteins from a single gene. Products of the four NFI genes differ in their abilities to either activate or repress transcription, likely through fundamentally different mechanisms. Here, we will review the properties of the NFI genes and proteins and their known functions in gene expression and development.