Fanconi anemia (FA) is a rare, mostly recessive, DNA repair deficiency disorder. Chromosomal instability is a cellular phenotype of FA, particularly when cells are exposed to DNA crosslinking agents. FA patients display developmental abnormalities, bone marrow failure (BMF), and predisposition to cancer including acute myeloid leukemia. Genetically, FA is a heterogeneous disease with 19 genes identified so far, and the encoded proteins participate in the FA/BRCA pathway that orchestrates repair of DNA damage caused by interstrand crosslinks. Eight FA proteins form a core complex and, along with FA associated proteins (FAAP), provide a key function that results in ubiquitination of two other FA proteins, FANCD2 and FANCI, a critical step in the pathway. But for FANCL and FANCT/UBE2T, biochemical functions of other Core proteins are yet unknown. Only a fraction of FA gene homologs are present in any invertebrate model organism, and thus zebrafish offers an opportunity to explore the functions of FA genes, particularly in understanding BMF, as hematopoiesis in zebrafish is well studied. We can knockout multiple genes in zebrafish with ease, so this allows exploring the consequence of loss of two or more genes that encode proteins known to interact. Except FANCS/BRCA1, homologs of all FA genes have been identified in zebrafish. Therefore, we used the CRISPR/Cas9 mediated targeted mutagenesis to generate loss of function mutants in fanca, fancb, fancc, fancd1/brca2, fancd2, fance, fancf, fancg, fanci, fancj/brip1, fancl, fancm, fancn/palb2, fanco/rad51c, fancp/slx4, fancq/ercc4, fanct/ube2t, and two FA-associated genes (faap100 and faap24). We designed two sgRNAs/gene and tested them for target-specific activity using our CRISPR-STAT method. We performed multiplexed mutagenesis by pooling the sgRNAs to FA genes based on their known interactions. We used our fluorescent PCR method to identify germline-transmitting founders with mutations in individual or multiple genes that were pooled together. We have begun the phenotypic characterization of all mutants. We will present data on the embryonic phenotypes for each of the genes when mutated individually and in combination with its interacting partners.