The transplantation of retinal pigment epithelial (RPE) cells derived from human iPS cells has been shown its effectiveness to age-related macular degeneration. However, the process of RPE differentiation takes a few months, and it could make inconsistent between batches. The combination of Yes-associated protein signaling activity which modulates ectodermal differentiation by cell density-dependent and variation in cell density in a vessel from operation might lead to this inconsistency. Therefore, we hypothesized the seeding operation that determines the position of each cell on a culture vessel plays an important role through RPE differentiation. In this research, we aim to stabilize the early phase of RPE differentiation derived from human iPS cells by mechanization of seeding operation. Human iPS cells (201B7 line) were seeded manually and mechanically on iMatrix-511-coated culture vessels and incubated with differentiation induction medium. At t = 24 h after seeding, two indices of spatial heterogeneity of cell density (local and global heterogeneity) were calculated by using nuclear stained images. In brief, local and global heterogeneity indicate the variation within each locality and the variation overall a culture vessel, respectively. The cells were collected on day 7 of differentiation and analyzed by flow cytometry to evaluate the ratio of neural ectoderm (Target lineage) and epidermal ectoderm (Non-target lineage). Based on spatial heterogeneity analysis, variation of global heterogeneity in machinery operation was lower than in manual operation. Then, the mean value of local heterogeneity in machinery operation was higher than in manual operation. Based on flow cytometric analysis, variation of the ratio of neural ectoderm and epidermal ectoderm in machinery operation was lower than manual operation. Moreover, the yield of neural ectoderm was improved by mechanization. It was suggested that the control of spatial heterogeneity by mechanization stabilized the early phase of RPE differentiation. In conclusion, we demonstrated that stabilization of the early phase of RPE differentiation derived from human iPS cells by mechanization of seeding operation.