Organotypic culture of human fetal testis has achieved fertilization-competent spermatids followed by blastocysts development. This study focuses on whether the organotypic culture of testicular tissue from infant boys with cryptorchidism could support the development of spermatogonia and somatic cells. Frozen-thawed tissues were cultured in two different media, with or without retinoic acid (RA), for 60 days and evaluated by tissue morphology and immunostaining using germ and somatic cell markers. During the 60-day culture, spermatocytes stained by boule-like RNA-binding protein (BOLL) were induced in biopsies cultured with RA. Increased AR expression ( p < 0.001) and decreased AMH expression ( p < 0.001) in Sertoli cells indicated advancement of Sertoli cell maturity. An increased number of SOX9-positive Sertoli cells ( p < 0.05) was observed, while the percentage of tubules with spermatogonia was reduced ( p < 0.001). More tubules with alpha-smooth muscle actin (ACTA, peritubular myoid cells (PTMCs) marker) were observed in an RA-absent medium ( p = 0.02). CYP17A1/STAR-positive Leydig cells demonstrated sustained steroidogenic function. Our culture conditions support the initiation of spermatocytes and enhanced maturation of Sertoli cells and PTMCs within infant testicular tissues. This study may be a basis for future studies focusing on maintaining and increasing the number of spermatogonia and identifying different factors and hormones, further advancing in vitro spermatogenesis.