Non-syndromic orofacial clefting (nsOFC) is among the most common congenital birth defects occurring up to 1 in 800 live births, with genetic and environmental causes. Genome wide association studies (GWAS) have identified several genetic loci that confer risk for nsOFC. However, more than half the heritable risk for nsOFC remains unknown and is considered ‘missing’. Moreover, continued sequencing of nsOFC patient DNA by whole exome sequencing and whole exome sequencing identify hundreds of single nucleotide polymorphism (SNPs). The identification of causal SNPs, however, continues to be a challenge in the OFC community. This is fueled partly by a lack of understanding of: (i) molecular mechanism and, (i) the gene regulatory network (GRN) governing differentiation of the relevant tissue, the embryonic superficial epithelia, also known as the periderm. Research has demonstrated that aberrant differentiation of the periderm, particularly the oral periderm results in pathological adhesions of surfaces within the developing oral cavity resulting in OFC. Further these adhesions can extend to the limbs which is a hallmarks feature in some forms of syndromic OFC (sOFC). In zebrafish, our model system of choice, knock-out of interferon regulatory factor 6 (irf6) ablated periderm marker expression and subsequently induces early embryonic lethality. The ortholog of IRF6 is a major genetic locus of Van der Woude syndrome (VWS) the most common form of sOFC and variants of IRF6 elevate risk for nsOFC. Therefore, we hypothesize that GRN of zebrafish periderm differentiation under the control of irf6 is a tool that can be used to identify novel OFC loci. Supporting this view, we have recently demonstrated that knock-down of an irf6 dependent gene encoding transcription factor Grainy-head like 3 (Grhl3) results in aberrant zebrafish periderm differentiation and GRHL3 was recently discovered as a novel VWS genetic locus. Hence it is likely that orthologs of genes encoding additional members of the periderm GRN harbor mutations in OFC patients. To identify cis–regulatory and transcriptional components in the periderm GRN, we performed: (i) a screen for periderm enhancers through in vivo green fluorescent protein (GFP) reporter assays, and, (ii) irf6 RNA-seq, followed by irf6 ChIP-seq to identify direct targets. From our screen for cis-regulatory elements we have identified a candidate human ZNF750 enhancer that directs GFP reporter expression in the zebrafish periderm. From our screen for irf6 direct targets we have identified several transcription factors including klf17, tfap2a and grhl3, all of which have variants in the human orthologs found in OFC patients. We further resolve the structure of the periderm differentiation GRN in zebrafish by assessing loss of function profiles for klf17, tfap2a and grhl3. Additionally, among the irf6 direct targets is a gene encoding another transcription factor, Zinc finger protein 750 (Znf750). We provide evidence to show that znf750 is expressed weakly in the zebrafish periderm. Further, we sequenced DNA in 500 nsOFC patient samples and identify a novel missense Ser160Pro ZNF750 variant which phenocopies the early embryonic lethality observed in irf6 mutants. Therefore, investigation of the zebrafish periderm GRN structure has facilitated the identification of OFC-associated risk loci.