Recent efforts aimed at integrating in vitro high-throughput screening (HTS) data into chemical toxicity assessments are necessitating increased understanding of concordance between chemical-induced responses observed in vitro versus in vivo. This investigation set out to (1) measure concordance between in vitro HTS data and transcriptomic responses observed in vivo, focusing on the liver, and (2) identify attributes that can influence concordance. Signal response profiles from 130 substances were compared between in vitro data produced through Tox21 and liver transcriptomic data through DrugMatrix, collected from rats exposed to a chemical for ≤5 days. A global in vitro-to-in vivo comparative analysis based on pathway-level responses resulted in an overall average percent agreement of 79%, ranging on a per-chemical basis between 41% and 100%. Whereas concordance amongst inactive chemicals was high (89%), concordance amongst chemicals showing in vitro activity was only 13%, suggesting that follow-up in vivo and/or orthogonal in vitro assays would improve interpretations of in vitro activity. Attributes identified to influence concordance included experimental design attributes (eg, cell type), target pathways, and physicochemical properties (eg, logP). The attribute that most consistently increased concordance was dose applicability, evaluated by filtering for experimental doses administered to rats that were within 10-fold of those related to likely bioactivity, derived using Tox21 data and high-throughput toxicokinetic modeling. Together, findings suggest that in vitro screening approaches to predict in vivo toxicity are viable particularly when certain attributes are considered, including whether activity versus inactivity is observed, experimental design, chemical properties, and dose applicability.