The aim of this study was to evaluate the impact of a professional development (PD) program on preschool teacher assistants' (TAs) attitudes toward multilingualism and self-efficacy in working with linguistically and culturally diverse children (LCDC) and their parents. The study was conducted in a northern peripheral city in Israel that reflects the country's multicultural, multilingual mosaic. Recently, as part of reforms in the city, PD for TAs was introduced to enrich the participants' practical knowledge about the development and education of linguistically and culturally diverse children. Qualitative and quantitative data were collected from 59 TAs before and after PD. Questionnaires focused on TAs' self-efficacy in multicultural classrooms, as well as on their attitudes toward multilingualism. In addition, participants reflected on an event in their preschool with a child whose home language was other than Hebrew--the societally dominant language--focusing on the TAs' emotional and behavioral responses. The qualitative and the quantitative data point to the impact of this PD program. Dependent variables t-test analysis revealed significant increase in TAs' self-efficacy to work with LCDC and with parents, thus supporting classroom multilingualism. Mixed-method analysis of the qualitative data showed a decrease in negative feelings such as anxiety, disappointment, low self-esteem, and unpreparedness for handling LCDC, and an increase in positive feelings such as happiness, pride, satisfaction, achievement, and self-efficacy. The discussion addresses three issues: the importance of multicultural, multilingual professional development for TAs, self-efficacy as a central characteristic of TAs' functioning, and the importance of TAs' relationships with parents.