Although methods of assisted reproduction are viable treatments for infertility, they are not always possible for azoospermic men. However, the use of stem cells may provide an alternative means of generating sperm for treatment. To date, primordial germ cell-like cells (PGCLCs) have been produced in culture from mouse and human pluripotent stem cells (PSCs). However, these cells cannot undergo later stages of spermatogenesis unless transplanted into the testes. The implication is that the development of functional sperm requires the testis niche, primarily established by Sertoli cells. In terms of a therapeutic solution, the generation of Sertoli cell-like cells (SCLCs) by PSC differentiation is one means of recreating the testis niche and inducing maturation among PGCLCs in vitro. Drawing from knowledge of gonadogenesis, this thesis demonstrates a method of generating SCLCs entirely in vitro. Mouse PSCs were sequentially induced to form intermediate mesoderm, SCLCs and steroidogenic cells by addition of growth factors BMP7 and FGF9, and small molecules TTNPB (a retinoic acid receptor agonist) and CHIR99021 (a GSK3 inhibitor). These cells were found to endogenously express transcription factors critical for male development. Over-expression of one factor in particular, Steroidogenic factor-1 (SF1), substantially increased induction efficiency. Intriguingly, conditioned medium derived from SCLCs was also capable of specifying germ cell-like cells from Epiblast-like cells (EpiLCs), possibly due to the presence of key inducer BMP4. Co-culture of SCLCs and EpiLCs resulted in the formation of 'testicular organoids' reminiscent of the seminiferous tubules. These organoids were even capable of upregulating meiotic marker Stra8 in the presence of retinoic acid, suggesting that subsequent stages of maturation may have been achieved. On the whole, this study constitutes a significant step towards the use of PSCs to recreate spermatogenesis and testicular cell interactions in vitro, and serves as a useful basis for further investigations into the role of SF1 in Sertoli cell differentiation and function.