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Spermatogonial differentiation is a process that commits germ cells to the complex process of spermatogenesis. Spermatogonial differentiation is mediated by the action of retinoic acid, which triggers major morphological and transcriptional changes. While these transcriptional changes have been well explored, there has been little effort devoted to epigenetic regulation surrounding spermatogonial development. This study aimed to uncover the timing and dynamics of chromatin organization during spermatogonial development within the context of these transcriptional changes. Using germ cell synchrony and the assay for transposase accessible chromatin and next generation sequencing (ATAC-seq) to isolate subpopulations of developing spermatogonia and identify accessible regions within their genome, we found that 50% of accessible regions in undifferentiated spermatogonia were condensed following retinoic acid action within 18 h. Surprisingly, genes with known functional relevance during spermatogonial development were accessible at all times, indicating that chromatin state does not impact transcription at these sites. While there was an overall decrease in gene accessibility during spermatogonial development, we found that transcriptionally active regions were not predictive of chromatin state.