The mitochondrial contribution to the maintenance of human embryonic stem cell (hESC) pluripotency and culture homeostasis remains poorly understood. Here, we sought to determine whether hESC adaptation to different feeder-free culture conditions is linked to a mitochondrial adaptation. The expression of ESC pluripotency factors and parameters of mitochondrial contribution including mitochondrial membrane potential, mtDNA content, and the expression of master mitochondrial genes implicated in replication, transcription, and biogenesis were determined in 8 hESC lines maintained in 2 distinct human feeders-conditioned media (CM): human foreskin fibroblast-CM (HFF-CM) and mesenchymal stem cell-CM (MSC-CM). We show a robust parallel trend between the expression of ESC pluripotency factors and the mitochondrial contribution depending on the culture conditions employed to maintain the hESCs, with those in MSC-CM consistently displaying increased levels of pluripotency markers associated to an enhanced mitochondrial contribution. The differences in the mitochondrial status between hESCs maintained in MSC-CM versus HFF-CM respond to coordinated changes in mitochondrial gene expression and biogenesis. Importantly, the culture conditions determine the mitochondrial distribution within the stage-specific embryonic antigen 3 positive (SSEA3(+)) and negative (SSEA3(-)) isolated cell subsets. hESC colonies in MSC-CM display an "intrinsic" high mitochondrial status which may suffice to support undifferentiated growth, whereas hESC colonies maintained in HFF-CM show low mitochondrial status, possibly relying on the production of autologous niche with higher mitochondrial status to support pluripotency and culture homeostasis. Pluripotency markers and mitochondrial status are concomitantly reverted on changing the culture conditions, supporting an unrecognized role of the mitochondria in response to hESC culture adaptation. We provide the first evidence supporting that hESCs adaptation to different feeder-free culture systems relies on a mitochondrial response.