Over the last 10 years, the U.S. federal Tox21 consortium has developed and evaluated in vitro HTS methods for hazard identification and mechanistic insight. Tox21 has generated data on thousands of pharmaceuticals and data-poor chemicals, characterized the limits and applications of in vitro methods, and enabled incorporation of HTS data into regulatory decisions. To more broadly address challenges in the field of toxicology, Tox21 released a new strategic and operational plan (Thomas et al. 2018) that expands the focus of its research activities. This plan proposed that Tox21 expand its portfolio of alternative test systems, address technical limitations of in vitro test systems, curate legacy in vivo toxicity testing data, establishing scientific confidence in in vitro test systems, and refine alternative methods for characterizing pharmacokinetics and in vitro assay disposition. The new Tox21 strategic and operational plan addresses key challenges to advance toxicology testing and will benefit both the organizations involved and the toxicology community.