During early seedling growth, the cotyledons transition from a storage tissue to become effectively the first leaf-like structures of the plant. In this programmed developmental process, they likely undergo a massive change in gene expression to redirect their metabolism and physiological processes. To define the developmental shifts in gene expression and begin to understand the gene regulatory networks that set this transition in motion, we carried out high-throughput RNA sequencing of cotyledons from seven developmental stages of soybean seedlings. We identified 154 gene models with high expression exclusively in the early seedling stages. A significant number (about 25 %) of those genes with known annotations were involved in carbohydrate metabolism. A detailed examination of glyoxylate cycle genes revealed the upregulation of their expression in the early stages of development. A total of approximately 50 % of the highly expressed genes whose expression peaked in the mid-developmental stages encoded ribosomal family proteins. Our analysis also identified 219 gene models with high expression at late developmental stages. The majority of these genes are involved in photosynthesis, including photosystem I- and II-associated genes. Additionally, the advantage of RNA-Seq to detect genes expressed at low levels revealed approximately 460 transcription factors with notable expression in at least one stage of the developing soybean seedling. Relatively over-represented transcription factor genes encode AP2, zinc finger, NAC, WRKY, and MYB families. These transcription factor genes may lead to the transcriptional reprogramming during the transition of seedling cotyledons from storage tissue to metabolically active organs that serve as the first functional leaves of the plant.