Nuclear Factor One (NFI) family of transcription factors regulate proliferation and multiple aspects of differentiation, playing analogous roles in embryonic development and various types of cancer. While all NFI family members are expressed in the developing brain and are involved in progression of brain cancers, their role in neuroblastoma has not been studied. Here we show that NFIB is required for the survival and proliferation of SHSY5Y neuroblastoma cells, assessed by viability and colony formation assays. Cdon, an Ig superfamily member, is a SHH dependence receptor that acts as a tumor suppressor in neuroblastoma. In the absence of NFI, Cdon is upregulated in the developing mouse brain, however the mechanisms by which its transcription is regulated remains unknown. We report CDON as a downstream target of NFIs in SH-SY5Y cells. There are three putative NFI binding sites within the one kb CDON promoter, two of which are occupied by NFIs in SH-SY5Y cells and human neural stem cells. In dual-luciferase assays, Nfib directly represses CDON proximal promoter activity. Moreover, silencing NFIB leads to upregulation of CDON in SH-SY5Y cells, however, decreased cell proliferation in NFIB silenced cells could not be rescued by concomitantly silencing CDON, suggesting other molecular players are involved. For instance, p21, an NFI target in glioblastoma and breast cancer cells, is also upregulated upon NFIB knock-down. We propose that NFIB is indispensable for SH-SY5Y cells which may involve regulation of apoptosis inducer proteins CDON and p21.