Telomeres are nucleoprotein structures at the ends of linear chromosomes, bound by various proteins. Some of these proteins protect the telomere ends, such as the shelterin complex, while others help regulate telomere length. Recently, our lab has characterized two telomere-binding proteins, HMBOX1 and ZBTB48, in mammalian cell lines. These proteins were found to be conserved across evolution and regulate telomere length. To understand their role at the organismal level, we have chosen to study these proteins in zebrafish, an emerging model organism for studying telomere biology. Due to a genome-wide duplication in teleost, Hmbox1 exists as paralogues in zebrafish. This study aims to investigate whether the telomere length regulatory functions of these proteins can be recapitulated in zebrafish and how they influence the organism. To facilitate the study, CRISPR-Cas9 knockout zebrafish lines and antibodies against each of these proteins were generated. However, the knockout fish lines did not exhibit any severe phenotypes, and there was no significant difference in the telomere length of the first generation of homozygous mutants compared to their wild-type counterparts. Conversely, this study has found more success in exploring the spatiotemporal expression of these proteins during early zebrafish development. Based on immunofluorescence staining, Hmbox1a was found to be expressed during the blastula and gastrulation stages (3 hpf to 10 hpf), while Hmbox1b is expressed in the notochord and pronephros of embryos from 9 hpf to 3 dpf. On the other hand, Zbtb48 was found to be highly expressed in a small population of cells in the gonads, specifically the germ stem cells (undergoing mitosis) and gametocytes (undergoing meiosis), according to the published scRNA-seq data. Additionally, Mtfp1 was consistently observed to be downregulated in the zbtb48-/- mutant, which was previously reported as one of the genes dysregulated by ZBTB48 in the human cell line study. Overall, although this study did not report any phenotypes related to the telomere function of these proteins, it has provided new information on their physiological spatiotemporal expression during development, which has not been described previously.