Spermatogenesis is a complex process where hormonal signals regulate the interaction of different cell types in a tight spatial and temporal fashion. The Senegalese sole (Solea senegalensis) is a marine flatfish that, in contrast to many fish, exhibits a semi-cystic, asynchronous pattern of spermatogenesis progression. This pattern is characterized by the release of spermatids into the tubule lumen, where they transform into spermatozoa. In this study, we used laser capture microdissection (LCM) to isolate cells from cysts containing spermatogonia, spermatocytes, spermatids or spermatozoa in order to investigate developmental patterns of gene expression. Furthermore, we also analyzed the stage-specific expression of the same set of genes throughout spermatogenesis (early-mid, late and maturing spermatogenic stages) in tissue fragments of the Senegalese sole testis. Genes analyzed by qPCR in cysts isolated by LCM and stage-specific testis samples included genes involved in steroid synthesis and action (3β-hsd, 17β-hsd, 20β-hsd, star, star-like, progesterone receptor), gonadotropin action (fshr, lhr), the kisspeptin system (kiss2, kiss2r) and other genes important for the production of mature gametes (zona pellucida 2.2, claudin and clusterin). Our results show that, in general, steroidogenesis-related genes tended to increase with spermatogenesis progression and that 3β-hsd and 20β-hsd were expressed in germ cells but 17β-hsd was not. Our results also show that fshr is expressed in most testicular cell types, including germ cells. In contrast, lhr is expressed only in late spermatogenesis and is not expressed in any of the germ cell types examined, indicating that, in contrast to fshr, lhr may be primarily expressed in non-germinal cells (e.g. Leydig cells). Furthermore, kisspeptin and its receptor were expressed in all germ cell types examined and, as expected, gamete maturation-related genes were more expressed in mature stages. These results illustrate that key factors that participate in the hormonal regulation of spermatogenesis in the Senegalese sole testis show complex cell type- and stage-specific patterns of gene expression.