Precise regulation over spermatogonial stem cell (SSC) function is integral for continuation of spermatogenesis. SSCs must balance self-renewal with the production of progenitor spermatogonia that are poised to enter into a pathway of differentiation, lest the self-renewing reservoir become exhausted, instigating azoospermic infertility. To expand our limited understanding of factors that drive self-renewal and maintenance of the SSC pool, we previously designed and implemented a high-throughput screening pipeline using a large-scale siRNA library in a mouse model. Preliminary data from this siRNA screen identified that knockdown of Chd4 (Chromodomain Helicase DNA Binding Protein 4); a member of the nucleosome remodelling and deacetylase complex, instigated a 50% reduction in putative SSCs in primary cultures of undifferentiated spermatogonia after 6 days. To validate these findings in vivo, we microinjected control or Chd4-knockdown spermatogonial populations into the testes of germ-cell ablated recipients, and assessed colonisation efficiency of the donor SSCs. In alignment with our preliminary findings, spermatogonial transplantation experiments revealed that knockdown of Chd4 significantly impaired SSC function, with the number of donor-derived colonies of spermatogenesis being reduced by 46%. Beyond these functional experiments, the expression profile of Chd4 was characterised using single cell RNA sequencing (scRNA-seq) of ‘whole testis’ (i.e. germ and somatic cells). These analyses revealed that expression levels are highest in the germ cell lineage; specifically in spermatogonia, and are most prominently elevated in SSCs. Finally, to explore mechanisms underlying the role of CHD4 in SSC maintenance, we used scRNA-seq to identify differentially expressed genes in ‘control’ versus ‘Chd4-knockdown’ populations. Findings revealed that CHD4 likely acts to repress expression of differentiation-driving genes, such as Sohlh2, while activating expression of genes important for maintenance of the undifferentiated state, such as Pten. These experiments are the first to depict a key role for CHD4 in SSC function and thus male fertility.