<p>The routine mass applications of human pluripotent stem (hPS) cell-derived progenies for regenerative medicine or high-throughput drug screenings will depend on the standardized supply of high-quality cells via controlled, efficient bioprocesses. Recent suspension culture (three-dimensional, 3D) strategies for hPS cell production in stirred-tank bioreactors (STBR) support this development. However, bioprocess inoculation still depends on adherent (two-dimensional, 2D) preculture, which is labor intensive, resource demanding and poorly controlled and limits process automation. Here we describe the controlled in-process production and dissociation of 3D cultured hPS cell aggregates directly in STBRs, tackling these challenges. The resulting cells can be used for the generation of high-density cryostocks, subsequently enabling the direct inoculation of 3D cultures, thereby entirely omitting the need for 2D preculture and the associated limitations. A key feature of this Protocol is the nonenzymatic, EDTA-based hPS cell aggregate dissociation approach in STBRs, enabling the impeller-based mechanical control of the dissociation process. The resulting cell suspension can be used for process reinoculation and seed train-based upscaling, as well as for the cryopreservation of produced hPS cells, ideally via controlled-rate freezing. Together, the protocol enables the efficient and flexible hPS cell suspension culture over multiple passages, maintaining karyotype stability and pluripotency. This Protocol can be easily implemented by any cell culture-educated scientist without extensive bioprocess training. Cell thawing requires 1 h, the 2D preculture requires 9 days, 2D cell passaging requires 1 h, bioreactor preparation requires 2 days, (direct) bioreactor inoculation requires 1.5 h, 3D STBR cultivation requires 3–4 days and STBR-based aggregate dissociation requires 2 h.</p>