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This mixed-methods study investigated how and in what ways preservice elementary teachers’ (n = 436) integrated STEM (iSTEM) teaching self-efficacy changed during their semester-long methods coursework. Participants attended three distinct institutions. Data sources included pre-post measures using the Self-Efficacy for Teaching Integrated STEM instrument, pre-post interviews with six participants (a total of 12 interviews), and demographic questions. Quantitative analysis using Analysis of Variance showed statistically significant positive gains in iSTEM teaching self-efficacy by the end of the semester. Qualitative content analysis revealed three sources of self-efficacy: mastery experiences, vicarious experiences, and physiological and affective states as major contributors toward preservice elementary teachers’ increased understanding and confidence in iSTEM teaching. Implications for STEM teacher preparation programs and future research are discussed.