The transcription factor GATA4 is found in Sertoli and Leydig cells, whereas SOX9 is exclusive to Sertoli cells, being both factors essential for the normal development of murine and human fetal testis. In turn, the steroidogenic acute regulatory protein (STAR) is specifically expressed in Leydig cells. Nevertheless, the function of STAR, GATA4 and SOX9 in peripubertal, adolescent and adult testes in Klinefelter syndrome and azoospermic patients remains poorly understood. To characterize the developmental expression of STAR, GATA4 and SOX9 in human testicular somatic cells, we performed immunofluorescence using fetal, peripubertal, adolescent and adult testes. Our findings demonstrate that STAR is absent in early fetal stages, but present in Leydig cells from 12 weeks of gestation, as well as in peripubertal, adolescent and adult Klinefelter patients, in the adult testis with idiopathic azoospermia and in men showing normal spermatogenesis. GATA4 was expressed in both Sertoli and Leydig cells during all the studied developmental stages and in peripubertal, adolescent and adult patients with and without spermatogenesis. SOX9 was mainly expressed in Sertoli cells in fetal, peripubertal, adolescent and adult Sertoli cell patients. In patients with Klinefelter syndrome as well as in men with or without spermatogenesis SOX9 was also found in Leydig cells. Our findings support the premise that STAR is a key steroidogenic protein for androgen development in the fetal testis, that GATA4 regulates Sertoli and Leydig cells during testis development and that SOX9 regulates the development of Sertoli cells and is present in the Leydig cells of patients with azoospermia.