As a model of early fetal brain development in Down syndrome, this study examines cortical organoids generated from isogenic trisomic and disomic iPSC lines. Initially pools of organoids from a trisomic versus disomic line found broad transcriptomic differences and modest differences in cell-type representation, suggesting a potential neurodevelopmental phenotype due to Trisomy 21. To better control for multiple sources of variation, we undertook a very robust study of ~1,200 organoids, using an expanded panel of six isogenic subclones (three disomic and three trisomic). The power of the experimental design was indicated by exceptionally strong detection of the ~1.5-fold difference in most chr21 genes. Despite some variability in secreted Aβ-40 levels between “identical” cell lines, this Alzheimer-related phenotype was detected as clearly correlated with Trisomy 21. However, the many statistically significant non-chr21 DEGs found in the small experiment fell away in the expanded study design, such that just three non-chr21 DEGs correlated to T21 status. Similarly, differences in cell-type representation of organoids varied somewhat between the six isogenic lines, but did not correlate with T21 status. Overall, our results indicate that even when organoid and batch variability are better controlled, common, subtle differences between isogenic cell lines (even subclones) may obscure, or be confused with, differences due to Trisomy 21. Interestingly, the neurodegenerative increase in Aβ due to T21 was strong enough to be evident in “fetal” organoids. In contrast, any neurodevelopmental phenotype that may be present in the ~2nd trimester of DS brain development may be more subtle, and within the range of variability in neurodifferentiation potential (unrelated to Trisomy 21) of our isogenic iPSC lines. The potential significance of two non-Chr21 DEGs that results suggest correlate with T21 is discussed.