Use of pro- (e.g., IL7 and IL15) and anti-inflammatory cytokines (e.g., IL4 and IL10) are being explored in cell therapy for their roles in directing and enhancing immunomodulation and survival of immune cells to alleviate conditions such as cancer and inflammatory disease, respectively. Due to their natural capacity to target inflamed and cancerous tissues, alongside their ability to surmount the clonality, expandability, and engineering hurdles linked to tissue-derived mesenchymal stem cells (MSCs), iPSC-derived MSCs (iMSCs) offer an optimal engineering platform for localized delivery of transgenic cytokines. Transgene expression, however, can be unstable or silenced during the differentiation of iPSCs to engineered iMSCs (EiMSCs). To ensure sustained transgene expression upon differentiation, we explored the use of ubiquitous chromatin-opening elements (UCOEs) in transgenic cassettes that express green fluorescent protein (GFP) under the EF1α promoter. Cassettes with and without a UCOE were inserted into the AAVS1 safe-harbor locus in iPSCs using mRNA encoding UltraSlice™ gene-editing endonucleases and single-stranded DNA (ssDNA) repair templates. Clonal iPSC lines were isolated by the single-cell deposition of GFP-positive cells, and biallelic insertion into AAVS1 was verified. Upon differentiation to EiMSCs, uniform GFP expression was only observed with a UCOE (>99% vs. ≤30% without) and persisted after 10 passages and 2 freeze-thaw cycles. We then applied this workflow to create pro- and anti-inflammatory EiMSC lines that are designed to overexpress the IL7-IL15 and IL4-IL10 fusion proteins, respectively. High knock-in efficiencies into iPSCs (~10%) enabled by the use of UltraSlice™ has allowed the establishment of clonal transgenic EiMSC lines without the use of a reporter system. EiMSCs stably express IL7-IL15 at 250 pg/mL, measured pre-passage in culture. These cell lines may prove promising in the development of therapies to treat a wide range of diseases.