Induced pluripotent stem cells (iPSCs) have significant potential for disease modeling and cell therapies. However, their wide-spread application has faced challenges, including batch-to-batch variabilities, and notable distinctions when compared to embryonic stem cells (ESCs). Some of these disparities can stem from using undefined culture conditions and the reprogramming procedure, however, the precise mechanisms remain understudied. Here, we compared gene expression data from over 100 iPSC and ESC lines cultivated under undefined and defined conditions. Defined conditions significantly reduced inter-PSC line variability, irrespective of PSC cell type, highlighting the importance of standardization to minimize PSC biases. This variability is concurrent with decreased somatic cell marker and germ layer differentiation gene expression and increased Ca