Strategies to improve the quality of hospital care are needed if ongoing gains in child health and survival are to be made. We previously reported on improvements in the quality of case management in hospitals following a guideline-based intervention in Lao PDR, with variation in the degree of change achieved between clinical conditions. This study aims to understand the factors that influenced the uptake of the guideline-based intervention, and its impact on care. This qualitative study was embedded in a mixed-methods evaluation of guideline implementation in nine hospitals in Lao PDR. Focus groups and individual interviews were conducted with 70 health staff from central, provincial, and district hospitals. The interview guide was based on the Theoretical Domains Framework. Inductive content analysis was performed on interview transcripts to identify themes, supported by field notes from the intervention. Findings were triangulated against previously reported quantitative outcomes using driver diagrams. Key influences on guidelines uptake related to the guideline and intervention (filling a void, physical accessibility, comprehensibility, training in guideline use), health staff (behavior regulation, trust in guidelines, and beliefs about consequences), and the environment (social influences particularly consensus and incorporation into clinical norms). The major barrier was family preference for treatments in conflict with guideline recommendations. This study identifies contextual factors that explain, as well as validate previously identified improvements in care following guideline implementation in Lao PDR. It provides novel understanding of why the same intervention may have a differential impact on different clinical conditions.