The primary spermatogonial stem cells (SSCs), which arise from gonocytes during neonatal development, serve as a foundational self-renewing reservoir to ensure continuous production of spermatozoa throughout adulthood. The transformation of gonocytes into SSCs takes place in a niche established by Sertoli cells. To date, the factors that guide Sertoli cells to establish the initial stem cell niche remain largely unknown. Using Sertoli cell-specific Arid4b knockout (Arid4bSCKO) mice, we demonstrated that ablation of ARID4B resulted in failure to establish a niche for the SSC formation. We performed RNA-Seq analysis to examine the gene expression profile of the Arid4bSCKO testes in comparison with that of control testes. Overall design: We extracted RNA from testes of the control and Arid4bSCKO mice at postnatal day 1.5 of age. Each genotypic group consisted of three pools of testes, and each pool contained four testes. Purified RNA was processed for RNA-Seq analysis using Illumina HiSeq 2500.