Testicular organoid models are tools to study testicular physiology, development, and spermatogenesis in vitro. However, few side-by-side comparisons of organoid generation method have been evaluated. Here, we directly tested whether the culture microenvironment is the prime determinant promoting testicular organoid self-assembly. Using Matrigel as a representative extracellular matrix (ECM), we compared multiple culture environments, 2D and 3D, ECM-free and ECM, for organoid self-assembly with immature murine testicular cells. De novo tissues were observed to self-assemble in all four culture environments tested within 72 h, however, these tissues only met requirements to be named organoids in 2D ECM and 3D ECM-free (3DF) culture methods. Based on these results, 3DF was selected for further study, and used to examine animal age as an independent variable. Organoid assembly was significantly delayed when using pubertal murine cells and entirely absent from adult murine and adult human cells. Organoid-conditioned medium and medium supplemented with 1% Matrigel did not improve organoid assembly in pubertal murine cells, but immature murine cells rescued the assembly of adult murine cells when cultured together as age-chimeric cell mixtures. In murine organoids cultured for 14 d, tubule-like structures exhibiting a highly biomimetic architecture were characterized, including some rare germ and spermatogonial stem cells. These structural organoids secreted high levels of testosterone and inhibin B over 12 weeks with preserved responsivity to gonadotropins. Collectively these studies, in which cellular self-assembly and organoid formation was achieved independent of the culture microenvironment, suggest that self-assembly is an innate property of immature testicular cells independent from, but capable of being promoted by, the culture environment. This study provides a template for studying testicular organoid self-assembly and endocrine function, and a platform for improving the engineering of functional testicular tissues.