Mobile instructional spaces such as retrofit buses, customized trucks or trailers, and repurposed shipping containers are innovative, unique venues for teaching and learning. STEM-focused mobile instructional spaces have emerged as vehicles for providing students and teachers with high-quality integrated STEM learning experiences that emphasize professional practices and 21st-century skills alongside a range of STEM content. In this article, we describe three best practices (creating a shared culture, fostering agency, and offering multiple pathways for engagement) for operating a STEM-focused mobile instructional space with K-12 students and teachers. We developed these best practices based on our own extensive work with two STEM-focused mobile instructional spaces, the BioBus and the MakerTruck. These practices have the potential to inform the work of formal and informal STEM educators who also operate mobile instructional spaces. Next steps for cementing the unique role STEM-focused mobile instructional spaces play in bridging the gap between in-school and out-of-school learning are discussed.